CAZypedia - User contributions [en-ca]

Carbohydrate Esterase Family 3

2020-12-02T19:40:22Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Stefen Stangherlin^^^
* [[Responsible Curator]]s: ^^^Michael Suits^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE3'''
|-
|'''Clan'''
|(α/β/α)-Sandwich
|-
|'''Mechanism'''

|Serine Hydrolase

|-
|'''Active site residues'''
|Known, Catalytic Triad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE3.html
|}
</div>


== Substrate specificities ==
Carbohydrate Esterase Family 3 is currently comprised entirely of de-''O''-acetylxylan esterases. Xylan is a plant cell-wall polysaccharide composed of β-1,4-linked xylose decorated with α-arabinofuranose and α-glucuronic acid substituents <cite>Faik2010</cite>.
== Catalytic Residues ==
Functionally characterized CE3 members are all known to contain the classical catalytic triad of Ser-His-Asp, typical of the SGNH hydrolase family of enzymes (see Fig. 1) <cite>Polgar2005 Molgaard2000</cite>. The active site residues are presented via four conserved consensus sequences (Blocks I-III and V), and have an altered nucleophilic “elbow” turn motif (-GxSxT- as opposed to the canonical -GxSxG- motif) compared to other related members of the α/β-hydrolase family <cite>UptonBuckley1995 Akoh2004</cite>. The catalytic triad along with the Block II Gly and Block III Asn residues that comprise the oxyanion hole, are universally conserved across all characterized CE3 enzymes. The Block V Asp residue mediates the amphoteric nature of the Block V His residue, which abstracts a proton from the Block I Ser to render it nucleophilic.[[Image:CE3_Figure.png|thumb|300px|Figure 1:''Tc''AE206 from ''Talaromyces cellulolyticus'' ([{{PDBlink}}5B5S PDB ID 5B5S]). Colours correspond to α-helices (cyan), β-sheets (magenta), loops (wheat), disulfide bond (yellow), calcium ion (orange) and the active site residues (green).]]

== Kinetics and Mechanism ==
CE3 esterases catalyze the hydrolysis of O-linked acetyl groups from xylan oligo- and poly-saccharides. The Block V His residue abstracts a proton from the Block I Ser, rendering it nucleophilic, which attacks the electrophilic carbonyl carbon of the acetyl group of the xylan substrate; generating a tetrahedral oxyanion intermediate that is stabilized by the backbone amides of the Block I Ser and Block II Gly, as well as the sidechain amide of the Block III Asn, together forming the oxyanion hole in the active site <cite>Uechi2016</cite>. Collapse of the oxyanion intermediate results in the formation of a transient acyl-enzyme intermediate and alcohol by-product <cite>Uechi2016</cite>. A hydrolytic water molecule is then deprotonated by the Block V His residue, and attacks the acyl-enzyme intermediate; hydrolyzing the bond and releasing acetate and the free enzyme <cite>Uechi2016</cite>. In the process, the Ser (Block I) is re-protonated and ready for another catalytic cycle.

The kinetics for enzymes from the CE3 family have been examined. For example, ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) was found to have a ''k''cat/''K''M of 2.5 and 1.2 mM-1s-1 for ''p''-nitrophenyl acetate (''p''NP-Ac) and acetylated xylan, respectively <cite>Correia2008</cite>. In other studies, ''Tc''AE206 ([{{PDBlink}}5B5S PDB ID 5B5S]) was not assayed against acetylated xylan, but the ''k''cat/''K''M with ''p''NP-Ac and ''p''-nitrophenyl butyrate (''p''NP-B) were reported as 44.7 and 4.1 mM-1s-1, respectively, while no activity was detected with ''p''‐nitrophenyl octanoate (''p''NP-O) as the substrate <cite>Watanabe2015</cite>.

== Three-dimensional structures ==
The CE3 family has a number of enzymes that have been structurally resolved. Examples include ''Tc''AE206 from ''Talaromyces cellulolyticus'' ([{{PDBlink}}5B5S PDB ID 5B5S]) (see Fig. 1) and ''Ct''Ces3-1 from ''Hungateiclostridium thermocellum'' (formerly ''Clostridium thermocellum'') ([{{PDBlink}}2VPT PDB ID 2VPT]). Both structures adopt an (α/β/α)-sandwich fold typical of the SGNH hydrolase family. The (α/β/α)-sandwich contains five central parallel β-strands forming a curved β-sheet, which is flanked by 5-6 α-helices <cite>Correia2008 Uechi2016</cite>. Additionally, both structures contain a calcium binding loop motif (DXVGX7DXn(D/N)) located above the N-terminal end of the central β-strand (β2) <cite>Watanabe2015</cite>. This binding motif is conserved across all currently characterized CE3s. A coordinated zinc ion was also observed next to a calcium ion in a ''Tc''AE206_S10A variant ([{{PDBlink}}5B5L PDB ID 5B5L]), however this was attributed to the use of ZnSO4 in the crystallization conditions <cite>Uechi2016</cite>. Unique to ''Tc''AE206 is a disulfide bond formed near the N-terminus (see Fig. 1) that is thought to position the catalytic Ser by stabilizing neighbouring areas, including a β-turn (β1) that involves the catalytic Ser <cite>Uechi2016 Watanabe2015</cite>.

== Family Firsts ==
;First characterized: In 1994, the sequence of XynB from ''Ruminococcus flavefaciens'' 17 was found to be related to family G xylanases <cite>Zhang1994</cite>. In 1997, BnaC from ''Neocallimastix patriciarum'' was found to have close relation to XynB and other enzymes known to be members of a diverse family of esterases <cite>Dalrymple1997</cite>. It wasn’t until 2000 that CesA from ''R. flavefaciens'' 17, which was shown to have significant sequence identity to XynB, was characterized with the ability to deacetylate acetylated xylans; thereby representing the first characterized enzymes of family 3 CEs <cite>Aurilia2000</cite>.
;First mechanistic insight: In 2000, CesA, XynB, and BnaC were aligned and shown to contain what was thought to be a Ser-His-Asp catalytic triad responsible for the observed esterase activity <cite>Aurilia2000</cite>. This was later confirmed by the structural resolution of ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) <cite>Correia2008</cite>.
;First 3-D structure: The first resolved structure was ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) from ''Hungateiclostridium thermocellum'' (formerly ''Clostridium thermocellum''), displaying the (α/β/α)-sandwich fold and Ser-His-Asp catalytic triad typical of SGNH hydrolases <cite>Correia2008</cite>.

== References ==
<biblio>
#Faik2010 pmid=20375115
#Polgar2005 pmid=16003488
#Molgaard2000 pmid=10801485
#UptonBuckley1995 pmid=7610479
#Akoh2004 pmid=15522763
#Uechi2016 pmid=27329813
#Watanabe2015 pmid=25825334
#Correia2008 pmid=18436237
#Zhang1994 pmid=7816035
#Dalrymple1997 pmid=9274014
#Aurilia2000 pmid=10846217
</biblio>

[[Category:Carbohydrate Esterase Families|CE003]]

Carbohydrate Esterase Family 3

2020-12-02T19:39:48Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Stefen Stangherlin^^^
* [[Responsible Curator]]s: ^^^Michael Suits^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE3'''
|-
|'''Clan'''
|(α/β/α)-Sandwich
|-
|'''Mechanism'''

|Serine Hydrolase

|-
|'''Active site residues'''
|Known, Catalytic Triad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE3.html
|}
</div>


== Substrate specificities ==
Carbohydrate Esterase Family 3 is currently comprised entirely of de-''O''-acetylxylan esterases. Xylan is a plant cell-wall polysaccharide composed of β-1,4-linked xylose decorated with α-arabinofuranose and α-glucuronic acid substituents <cite>Faik2010</cite>.
== Catalytic Residues ==
Functionally characterized CE3 members are all known to contain the classical catalytic triad of Ser-His-Asp, typical of the SGNH hydrolase family of enzymes (see Fig. 1) <cite>Polgar2005 Molgaard2000</cite>. The active site residues are presented via four conserved consensus sequences (Blocks I-III and V), and have an altered nucleophilic “elbow” turn motif (-GxSxT- as opposed to the canonical -GxSxG- motif) compared to other related members of the α/β-hydrolase family <cite>UptonBuckley1995 Akoh2004</cite>. The catalytic triad along with the Block II Gly and Block III Asn residues that comprise the oxyanion hole, are universally conserved across all characterized CE3 enzymes. The Block V Asp residue mediates the amphoteric nature of the Block V His residue, which abstracts a proton from the Block I Ser to render it nucleophilic.[[Image:CE3_Figure.png|thumb|300px|Figure 1:''Tc''AE206 from ''Talaromyces cellulolyticus'' ([{{PDBlink}}5B5S PDB ID 5B5S]). Colours correspond to α-helices (cyan), β-sheets (magenta), loops (wheat), disulfide bond (yellow), calcium ion (orange) and the active site residues (green).]]

== Kinetics and Mechanism ==
CE3 esterases catalyze the hydrolysis of O-linked acetyl groups from xylan oligo- and poly-saccharides. The Block V His residue abstracts a proton from the Block I Ser, rendering it nucleophilic, which attacks the electrophilic carbonyl carbon of the acetyl group of the xylan substrate; generating a tetrahedral oxyanion intermediate that is stabilized by the backbone amides of the Block I Ser and Block II Gly, as well as the sidechain amide of the Block III Asn, together forming the oxyanion hole in the active site <cite>Uechi2016</cite>. Collapse of the oxyanion intermediate results in the formation of a transient acyl-enzyme intermediate and alcohol by-product <cite>Uechi2016</cite>. A hydrolytic water molecule is then deprotonated by the Block V His residue, and attacks the acyl-enzyme intermediate; hydrolyzing the bond and releasing acetate and the free enzyme <cite>Uechi2016</cite>. In the process, the Ser (Block I) is re-protonated and ready for another catalytic cycle.

The kinetics for enzymes from the CE3 family have been examined. For example, ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) was found to have a ''k''cat/''K''M of 2.5 and 1.2 mM-1s-1 for ''p''-nitrophenyl acetate (''p''NP-Ac) and acetylated xylan, respectively <cite>Correia2008</cite>. In other studies, ''Tc''AE206 ([{{PDBlink}}5B5S PDB ID 5B5S]) was not assayed against acetylated xylan, but the ''k''cat/''K''M with ''p''NP-Ac and ''p''-nitrophenyl butyrate (''p''NP-B) were reported as 44.7 and 4.1 mM-1s-1, respectively, while no activity was detected with ''p''‐nitrophenyl octanoate (''p''NP-O) as the substrate <cite>Watanabe2015</cite>.

== Three-dimensional structures ==
The CE3 family has a number of enzymes that have been structurally resolved; ''Tc''AE206 from ''Talaromyces cellulolyticus'' ([{{PDBlink}}5B5S PDB ID 5B5S]) (see Fig. 1) and ''Ct''Ces3-1 from ''Hungateiclostridium thermocellum'' (formerly ''Clostridium thermocellum'') ([{{PDBlink}}2VPT PDB ID 2VPT]). Both structures adopt an (α/β/α)-sandwich fold typical of the SGNH hydrolase family. The (α/β/α)-sandwich contains five central parallel β-strands forming a curved β-sheet, which is flanked by 5-6 α-helices <cite>Correia2008 Uechi2016</cite>. Additionally, both structures contain a calcium binding loop motif (DXVGX7DXn(D/N)) located above the N-terminal end of the central β-strand (β2) <cite>Watanabe2015</cite>. This binding motif is conserved across all currently characterized CE3s. A coordinated zinc ion was also observed next to a calcium ion in a ''Tc''AE206_S10A variant ([{{PDBlink}}5B5L PDB ID 5B5L]), however this was attributed to the use of ZnSO4 in the crystallization conditions <cite>Uechi2016</cite>. Unique to ''Tc''AE206 is a disulfide bond formed near the N-terminus (see Fig. 1) that is thought to position the catalytic Ser by stabilizing neighbouring areas, including a β-turn (β1) that involves the catalytic Ser <cite>Uechi2016 Watanabe2015</cite>.

== Family Firsts ==
;First characterized: In 1994, the sequence of XynB from ''Ruminococcus flavefaciens'' 17 was found to be related to family G xylanases <cite>Zhang1994</cite>. In 1997, BnaC from ''Neocallimastix patriciarum'' was found to have close relation to XynB and other enzymes known to be members of a diverse family of esterases <cite>Dalrymple1997</cite>. It wasn’t until 2000 that CesA from ''R. flavefaciens'' 17, which was shown to have significant sequence identity to XynB, was characterized with the ability to deacetylate acetylated xylans; thereby representing the first characterized enzymes of family 3 CEs <cite>Aurilia2000</cite>.
;First mechanistic insight: In 2000, CesA, XynB, and BnaC were aligned and shown to contain what was thought to be a Ser-His-Asp catalytic triad responsible for the observed esterase activity <cite>Aurilia2000</cite>. This was later confirmed by the structural resolution of ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) <cite>Correia2008</cite>.
;First 3-D structure: The first resolved structure was ''Ct''Ces3-1 ([{{PDBlink}}2VPT PDB ID 2VPT]) from ''Hungateiclostridium thermocellum'' (formerly ''Clostridium thermocellum''), displaying the (α/β/α)-sandwich fold and Ser-His-Asp catalytic triad typical of SGNH hydrolases <cite>Correia2008</cite>.

== References ==
<biblio>
#Faik2010 pmid=20375115
#Polgar2005 pmid=16003488
#Molgaard2000 pmid=10801485
#UptonBuckley1995 pmid=7610479
#Akoh2004 pmid=15522763
#Uechi2016 pmid=27329813
#Watanabe2015 pmid=25825334
#Correia2008 pmid=18436237
#Zhang1994 pmid=7816035
#Dalrymple1997 pmid=9274014
#Aurilia2000 pmid=10846217
</biblio>

[[Category:Carbohydrate Esterase Families|CE003]]

Carbohydrate Esterase Family 3

2020-12-02T19:39:04Z

Joel Weadge: /* Kinetics and Mechanism */

Glycoside Hydrolase Family 105

2020-12-02T19:36:57Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}

* [[Author]]: ^^^James Stevenson^^^
* [[Responsible Curator]]: ^^^Joel Weadge^^^

----


<div style="float: right">
{| {{Prettytable}}
|-
| {{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH105'''
|-
| '''Clan'''
| none
|-
| '''Mechanism'''
| N/A
|-
| '''Active site residues'''
| known
|-
| {{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH105.html
|}
</div>



== Substrate specificities ==
GH105 enzymes are a class of unsaturated glucuronyl/galacturonyl hydrolases found mainly in bacteria, but a few fungal and a handful of archaeal enzymes have also been annotated <cite>Cantarel2009</cite>. Much like the [[Glycoside Hydrolase Family 88]], enzymes from GH105 perform hydrolysis via a hydration of the double bond between the C-4 and C-5 carbons of the terminal monosaccharide of their substrates <cite>Munoz-Munoz2017 Jongkees2011</cite>. Enzymes from GH105 have been organized into three subgroups: unsaturated rhamnogalacturonidases, D-4,5-unsaturated β-glucuronyl hydrolases, and D-4,5-unsaturated α-galacturonidases. The unifying feature shared between these substrates is the presence of the non-reducing monosaccharide 4-deoxy-L-threo-hex-4-enopyranuronosyl that binds at the -1 active site of the enzymes and is linked to the +1 sugar via its anomeric C-1 carbon. The 4-deoxy-L-threo-hex-4-enopyranuronosyl saccharide can be inferred as ΔGalA or ΔGlcA depending on whether it assumes an α- or β- configuration, respectively, at the anomeric C-1 carbon. In degradable substrates, the sugar present at the +1 position can be linked via its C-2, C-4, or C-6 carbon, given the substrate preference of individual enzymes <cite>Zhang2009 Munoz-Munoz2017</cite>. Some of the various carbohydrate sources targeted by GH105 enzymes include: rhamnogalacturonan-I, ulvan, and the arabinogalactan decoration on certain cell wall proteins <cite>Itoh2006 Itoh2006-1 Collen2014 Munoz-Munoz2017</cite>.

== Kinetics and Mechanism ==
GH105 enzymes do not act via a typical Koshland retaining or inverting mechanism <cite>Koshland1953</cite>, rather the current proposed mechanism of action for these enzymes is hydrolysis through syn-hydration of the double bond between the C-4 and C-5 carbons of the enopyranuronosyl residue of their substrate <cite>Itoh2006</cite>. This hydration reaction forms a hemiketal that undergoes spontaneous rearrangement to form an intermediate hemiacetyl, which undergoes further rearrangement resulting in the breakage of the bond to the neighbouring saccharide (at the +1 subsite of the enzyme) of the polymer. This mechanism was initially theorized based on the oligosaccharide and amino acid arrangement in a substrate-bound crystal structure <cite>Itoh2006-1</cite>, but has been confirmed through kinetic isotope effects and NMR analysis in the highly related unsaturated glucuronyl hydrolase family [[GH88]] <cite>Jongkees2011 Jongkees2014</cite>.

The kinetics for enzymes from the GH105 family have been determined. Specifically, YteR from ''Bacillus subtilis'' was found to have a ''k''cat and ''K''M of 0.2±0.011 s-1 and 100±14 μM , respectively, against the substrate ΔGalA-Rha. In contrast, YesR from ''B. subtilis'' was found to have much higher values for both these kinetic parameters, 13.9±0.7 s-1 and 719±75 μM for ''k''cat and ''K''M, respectively, with the same substrate <cite>Itoh2006-1</cite>. BT3687 from ''B. thetaiotaomicron'' was determined to have a ''k''cat of 0.59±0.057 s-1 and a ''K''M of 71.87±12.51 μM against the substrate ΔGlcA-GlcNAc <cite>Munoz-Munoz2017</cite>.

Although it is atypical for a glycoside hydrolase family to contain enzymes capable of degrading both α- or β-linked substrates, this has also been observed in other families that deviate significantly from typical acid-base mechanisms (''e.g.'' [[GH3]], [[GH4]]) <cite>Rye2000</cite>.

== Catalytic Residues ==
A single aspartate residue has been proposed to be responsible for the hydration reaction based on substrate-complexed X-ray crystal structures, sequence conservation, and site-directed mutagenesis <cite>Itoh2006 Itoh2006-1</cite>. The first enzyme classified into the GH105 family (YteR; [{{PDBlink}}1NC5 PDB ID 1NC5] from ''B. subtilis'') was originally predicted to be a lyase based on 65% amino acid sequence similarity and over 60% matching secondary-structure characteristics with an ''N''-acyl-D-glucosamine 2-epimerase <cite>Zhang2009</cite>. Following sequence comparison to a GH88 hydrolase (a ''B. subtilis'' UGL enzyme), and additional functional characterization, YteR was determined to possess unsaturated galacturonyl hydrolase activity <cite>Itoh2006-1</cite>. A conserved aspartate residue (D143), was found to be the most likely candidate for initiating the hydration reaction, while a second conserved residue, histidine (H189), serves to correctly position a water molecule for deprotonation and addition to the C-5 carbon of the monosaccharide in the enzyme's -1 subsite <cite>Itoh2006-1</cite>. Based on sequence alignment and structural analysis, an arginine residue may take the place of this histidine residue in some GH105 enzymes (e.g. [{{PDBlink}}4CE7 PDB ID 4CE7] and [{{PDBlink}}5NOA PDB ID 5NOA]) <cite>Pettersen2004 Collen2014 Munoz-Munoz2017</cite>.

== Three-dimensional structures ==
A number of crystal structures of GH105 unsaturated glucuronyl hydrolases expressed in bacteria have been solved, including several structures from ''B. subtilis'' <cite>Zhang2009 Itoh2006 Itoh2006-1</cite>([{{PDBlink}}1NC5 PDB ID 1NC5]), and ''B. thetaiotaomicron'' <cite>Munoz-Munoz2017 Collen2014</cite>([{{PDBlink}}3K11 PDB ID 3K11]), as well as one each from ''Bacteriodes vulgatus'' ([{{PDBlink}}4Q88 PDB ID 4Q88]), ''Clostridium acetobutylicum'' <cite>Germane2015</cite>, ''Klebsiella pneumoniae'' ([{{PDBlink}}3PMM PDB ID 3PMM]), and ''Salmonella enterica'' ([{{PDBlink}}3QWT PDB ID 3QWT]). A single enzyme from the fungus ''Thielavia terrestris'' has also been solved ([{{PDBlink}}4XUV PDB ID 4XUV]). All of these enzymes share an (α/α)6-barrel structure (also similar to that of the related GH88 enzymes), with the main differences being seen in the structure of the loop region that determines the architecture of the binding site. At the bottom of the active site pocket is a conserved WxRxxxW motif, with the tryptophan and arginine residues forming a pocket that engages the carboxyl group on the ΔGalA/GlcA monosaccharide of the -1 subsite <cite>Itoh2006</cite>. While several residues may be conserved in sequence and position at the -1 subsite, the +1 subsite is much more variable, which likely accounts for the ability of this enzyme family to catalyze the hydrolysis of polysaccharides containing α- or β-bonds linked to the C-2, -4, or -6 carbon of the +1 saccharide <cite>Collen2014</cite>.

== Family Firsts ==
; First stereochemistry determination: Crystal structure of substrate-bound ''B. subtilis'' YteR unsaturated rhamnogalacturonan hydrolase in 2006 ([{{PDBlink}}1NC5 PDB ID 1NC5]). Functional analysis of this enzyme detected loss of a C=C bond compared to a detectable increase in α-keto acid following enzyme-substrate incubation <cite>Itoh2006-1</cite>.
; First catalytic residue identification: Crystal structure analysis of the ''B. subtilis'' YteR enzyme complexed with a ΔGlc-GalNac substrate analog suggested Asp143 is responsible for initiating the hydration reaction and kinetic assessment of a D143N mutant of YteR showed complete loss of catalytic activity <cite>Itoh2006-1</cite>.
; First evidence of hydration-based mechanism: While the mechanism of GH105 enzymes has not been fully described, the mechanism of the unsaturated glucuronyl hydrolase (UGL) from ''Clostridium perfringens'' (a closely-related GH88 protein) was determined via NMR using a methyl ketal intermediate analogue and monitoring of the reaction product during enzyme-substrate incubation in D2O <cite>Jongkees2011</cite>.
; First 3-D structure: The 1.6Å crystal structure of the ''B. subtilis'' protein YteR ([{{PDBlink}}1NC5 PDB ID 1NC5]), initially predicted to be a lyase-type enzyme, was reported in 2005 <cite>Zhang2009</cite>.

== References ==
<biblio>
#Cantarel2009 pmid=18838391
#Munoz-Munoz2017 pmid=28637865
#Jongkees2011 pmid=22047074
#Zhang2009 pmid=15906318
#Itoh2006 pmid=16870154
#Itoh2006-1 pmid=16781735
#Collen2014 pmid=24407291
#Koshland1953 Koshland, D.E. (1953) Stereochemistry and the Mechanism of Enzymatic Reactions. ''Biological Reviews'', vol. 28, no. 4., pp. 416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x].
#Jongkees2014 pmid=24573682
#Rye2000 pmid=11006547
#Pettersen2004 pmid=15264254
#Germane2015 pmid=26249707

</biblio>

[[Category:Glycoside Hydrolase Families|GH105]]

Glycoside Hydrolase Family 52

2020-12-02T19:28:51Z

Joel Weadge: /* Substrate specificities */

{{CuratorApproved}}
* [[Author]]: ^^^Julie Grondin^^^ and ^^^Brian Lowrance^^^
* [[Responsible Curator]]: ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH52'''
|-
|'''Clan'''
|GH-O
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH52.html
|}
</div>


== Substrate specificities ==
The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are generally monospecific, functioning as ''exo''-β-xylosidases (EC [{{EClink}}3.2.1.37 3.2.1.37]) that cleave the terminal xylose residues from the non-reducing end of artificial xylosides and xylooligosaccharides (e.g., ''p''NP-β-D-xylopyranoside <cite>Bravman2001, Espina2014</cite>, xylobiose <cite>Espina2014</cite>, and xylotriose <cite>Espina2014</cite>). Low levels of α-L-arabinofuranoside activity has also been observed within members of the GH52 family <cite>Suzuki2014, Bravman2003a</cite>, which is similar to the specificity noted for [[GH13]] and [[GH54]] for β-xylooligosaccharides and α-L-arabinofuranosides. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site <cite>Lee2007, Utt1991</cite>. Under certain conditions, some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has also been infrequently observed in other [[glycoside hydrolase]]s <cite>Romero2019</cite>. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity <cite>Huang2014</cite> and transition from a [[glycoside hydrolase]] to a glycosynthase <cite>Dann2014</cite> has been achieved.

== Kinetics and Mechanism ==
Retention of stereochemistry has been observed in GH52 β-xylosidases, which is characteristic of a classical [[Koshland double-displacement mechanism]] <cite>Koshland1953</cite>. This was first determined by Bravmen and coworkers using 1H-NMR to analyze the breakdown products of ''p''NP-β-D-xylopyranoside by XynB2, a β-xylosidase from ''Bacillus stearothermophilus'' T-6 <cite>Bravman2001</cite>. Further detailed analysis within this family was published in 2003 on the ''B. stearothermophilus'' XynB2 enzyme, which contained pH dependence studies (enzymatic catalysis is dependent on ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and kinetic analyses (''p''NP-xylobiose ''k''cat/''K''M of 140 s-1mM-1; xylobiose and xylotriose ''K''M values of 17.1x104 M-1 and 9.6x104 M-1, respectively) <cite>Bravman2003a</cite>.

== Catalytic Residues ==
Site-directed mutagenesis, chemical rescue, and kinetic profiling of XynB2 from ''B. stearothermophilus'' T-6 identified E335 as the [[catalytic nucleophile]] and D495 as the [[general acid/base]] <cite>Bravman2001, Bravman2003b</cite>. The catalytic nucleophile (E335) is conserved within the WVVNEGEY motif, which is found approximately 150 residues up-stream from the EITTYDSLD motif containing the general acid/base (D495). These results were further confirmed following the structural analysis of a GH52 from ''Geobacillus thermoglucosidasius'' <cite>Espina2014</cite>, where in this structure the 6.5 Å separation of Glu and Asp in the active site was typical of retaining enzymes.

== Three-dimensional structures ==
[[File:Figure1_dimer.PNG|400px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]
Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (α/α)6 barrel domain (Figure 1) that has led to their classification into [[Clan]] GH-O, together with [[GH116]]. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy 4H3 half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-β-xylosidase activity, while inhibiting activity on large polymers, such as xylan.

== Family Firsts ==
;First stereochemistry determination: XynB2 from ''Bacillus stearothermophilus'' T-6 by 1H-NMR for the hydrolysis of ''p''NP-β-D-xylopyranoside <cite>Bravman2001</cite>.
;First catalytic nucleophile identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis and chemical rescue <cite>Bravman2003</cite>.
;First general acid/base residue identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis, chemical rescue, and pH profiling <cite>Bravman2003</cite>.
;First 3-D structure: GT2_24_00240 from ''Geobacillus thermoglucosidasius'' NBRC 107763 <cite>Espina2014</cite>.

== References ==

<biblio>
#Bravman2001 pmid=11322943
#Espina2014 pmid=24816105
#Suzuki2001 pmid=11330658
#Bravman2003a pmid=12950180
#Lee2007 pmid=18051350
#Utt1991 pmid=1905520
#Huang2014 pmid=24122394
#Dann2014 pmid=25484225
#Romero2019 pmid=31024890
#Bravman2003b pmid=12738774
#Koshland1953 Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]

</biblio>

[[Category:Glycoside Hydrolase Families|GH052]]

Carbohydrate Esterase Family 2

2020-12-01T19:25:59Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from ''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'' and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 over position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of its β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:24:46Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from ''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'' and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 over position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:24:23Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'' and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 over position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:23:38Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) ''Cellvibrio japonicus'' and and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 over position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:20:38Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 over position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:18:29Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:17:41Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:15:42Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:12:13Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine then acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:10:29Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes involves a catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T19:08:23Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:55:08Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2, ''Cj''CE2B, and Est2A contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:52:49Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2, ''Cj''CE2B, and Est2A contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:50:19Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad, as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:40:55Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad, as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad<cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:39:59Z

Joel Weadge: /* Substrate specificities */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad<cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-12-01T17:36:34Z

Joel Weadge: /* Substrate specificities */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members preferentially deacetylate xylopyranosides at positions 3 and 4 over the 2 position. These enzymes have also demonstrated deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position. Given the greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad<cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:39:50Z

Joel Weadge:

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:35:54Z

Joel Weadge:

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known, Catalytic Dyad
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:32:33Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:31:54Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:31:18Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:29:49Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:29:06Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:27:13Z

Joel Weadge: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-26T20:25:01Z

Joel Weadge:

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to rule. It has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T20:02:31Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets are shown in cyan and magenta as cartoon models, respectively.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T20:01:29Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets are shown in cyan and magenta as cartoon models, respectively.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(Figure 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:58:28Z

Joel Weadge: /* Family Firsts */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:57:44Z

Joel Weadge: /* Family Firsts */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as, ''Ct''CE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:55:18Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:54:36Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:53:46Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:52:40Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:51:24Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel that form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:48:16Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five β-strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:47:37Z

Joel Weadge: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:45:34Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:44:26Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:43:15Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M values of 2.01, 0.71, 0.38 and 3.13 s-1 µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1 µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1 µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1 µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:42:55Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''cat/''K''M of 2.01, 0.71, 0.38 and 3.13 s-1 µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''cat/''K''M value of 2.33 x 10-3 s-1 µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''cat/''K''M values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''cat/''K''M values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1 µM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''cat/''K''M values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1 µM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:39:44Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed kcat/KM of 2.01, 0.71, 0.38 and 3.13 s-1×µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a kcat/KM value of 2.33 x 10-3 s-1×µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased kcat/KM values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed kcat/KM values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the kcat/KM values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:23:58Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and influences a water molecule so that it can attack the ester of the acetyl group attached to the catalytic serine; thereby generating a new tetrahedral intermediate state, which is also stabilized by the residues of the oxyanion hole. Upon release of the acetyl group, the histidine aids in donating a proton to the serine to restore the catalytic site to its original state <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed kcat/KM of 2.01, 0.71, 0.38 and 3.13 s-1×µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a kcat/KM value of 2.33 x 10-3 s-1×µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased kcat/KM values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed kcat/KM values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the kcat/KM values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:23:31Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and influences a water molecule so that it can attack the ester of the acetyl group attached to the catalytic serine; thereby generating a new tetrahedral intermediate state, which is also stabilized by the residues of the oxyanion hole. Upon release of the acetyl group, the histidine aids in donating a proton to the serine to restore the catalytic site to its original state <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed kcat/KM of 2.01, 0.71, 0.38 and 3.13 s-1×µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a kcat/KM value of 2.33 x 10-3 s-1×µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased kcat/KM values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed kcat/KM values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the kcat/KM values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]

Carbohydrate Esterase Family 2

2020-11-16T19:23:03Z

Joel Weadge: /* Kinetics and Mechanism */

{{CuratorApproved}}
* [[Author]]: ^^^Bobby Lamont^^^
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
|-
|'''Clan'''
|α/β-hydrolase
|-
|'''Mechanism'''
|Serine Hydrolase
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CE2.html
|}
</div>


== Substrate specificities ==

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.

== Catalytic Residues ==

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.

== Kinetics and Mechanism ==
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate, and a serine-substrate tetrahedral intermediate is stabilized by the residues of the oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and influences a water molecule so that it can attack the ester of the acetyl group attached to the catalytic serine; thereby generating a new tetrahedral intermediate state, which is also stabilized by the residues of the oxyanion hole. Upon release of the acetyl group, the histidine aids in donating a proton to the serine to restore the catalytic site to its original state <cite>Alalouf2011</cite>.

The characterized enzymes were all tested using ''p''NP-Ac, which showed kcat/KM of 2.01, 0.71, 0.38 and 3.13 s-1×µM-1 for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a kcat/KM value of 2.33 x 10-3 s-1×µM-1 showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased kcat/KM values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed kcat/KM values of 7.33 x 10-5, 7.67 x 10-4, and 2.33 x 10-4 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the kcat/KM values were 9.67 x 10-4, 6.5 x 10-4, and 2.68 x 10-2 s-1xµM-1 for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite>

== Three-dimensional structures ==

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.

The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.

== Family Firsts ==
;First characterized:
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.

;First mechanistic insight:
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.
;First 3-D structure:
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.

== References ==
<biblio>
#Montanier2009 pmid=19338387
#Dalrymple1997 pmid=9274014
#Hall1988 pmid=3066698
#Till2013 pmid=23345031
#Topakas2010 pmid=19968989
#Bayer2004 pmid=15487947
#Alalouf2011 pmid=21994937
</biblio>

[[Category:Carbohydrate Esterase Families|CE002]]