CAZypedia - User contributions [en-ca]

Polysaccharide Lyase Family 7

2020-06-17T12:25:27Z

Jan-Hendrik Hehemann:

{{CuratorApproved}}
* [[Author]]s: ^^^Nadine Gerlach^^^
* [[Responsible Curator]]: ^^^Jan-Hendrik Hehemann^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Polysaccharide Lyase Family PL7'''
|-
|'''3D Structure'''
|β jelly roll
|-
|'''Mechanism'''
|β-elimination
|-
|'''Active site residues'''
|R, Q, H, 2xY
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}PL7.html
|}
</div>


== Mechanism ==
[[Image:AlyPL7 MultipleSequenceAlignment.JPG|thumb|400px|'''Figure 1. Multiple protein sequence alignment of Aly PL7 ''' as well as a secondary structure prediction with the crystallized PL7 from ''Klabsiella pneumoniae'' ([{{PDBlink}}4OZX PDB ID 4OZX]). Conserved residues in the homologues are colored in red and (putative) catalytic residues are indicated by a star. The multiple protein sequence alignment was done with Espript3.0 <cite>Robert2014</cite>.]]
Alginate lyases (Alys) of all families, PL5-7, PL14-15, PL17-18, catalyze the depolymerization of alginate in three steps: (I) removal of the negative charge on the carboxylate anion, (II) general base-catalyzed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond <cite>Gacesa1986</cite>. Most PL7s are endo-active, i.e. acting within a poly- or oligosaccharide and releasing smaller alginate fragments. Some PL7 are exo-acting cleaving a monosaccharide from the non-reducing end of the polymer <cite>Thomas2013</cite>. In both modes of action, a new non-reducing end with a 4-deoxy-L-erythro-hex-4-en pyranosyl uronate residue (Δ) is formed.

Some marine PL7 alginate lyases require a calcium cation for substrate recognition and binding <cite>Thomas2013</cite>. Ca<sup>2+</sup> is weakening the ionic interactions between substrate (polyanion) and PL7 (polycation) by reducing the surface density of the alginate, charge and therefore increasing the enzyme activity <cite>Favorov1979</cite>. However, there have also been reports where mono- or divalent (metal) cations did not increase or even decreased enzymatic activity <cite>Jagtap2014 Badur2015</cite>. Therefore, it is not clear if Ca<sup>2+</sup> is required for recognition and / or binding. However, one could speculate if cations like Ca<sup>2+</sup> are creating a more accessible form of alginate. In the presence of divalent cations, the confirmation of alginate is changed due to cross linkages with guluronate residues from opposing chains forming a so-called egg box model <cite>Grant1973</cite>. Thereby, the 3D confirmation of alginate is changed, forming hydrogels which might be an easier target in the marine environment as its dissolved form.

== Kinetics and catalytic residues ==
Several structural and biochemical analyses of wild type and mutated PL7s revealed five residues forming the active site: arginine (R), glutamine (Q), histidine (H), tyrosine (Y) <cite>Preston2000 Yamasaki200 Yamasaki2005</cite>, which are present in three highly conserved regions: R*ELR*ML, VIIGQ(I/V)H, YFKAG*Y*Q respectively (Figure 1) <cite>Wong2000</cite>. It has been proposed that in PL7 ALY-1 from ''Corynebacterium'' sp. Q117+Y195 interact near the reaction site of alginate to maintain proper orientation of the substrate, R72 interacts with alginate due to the formation of salt bridges with the carboxyl groups at the C5 and H119 acts as a base to deprotonate <cite>Osawa2015</cite>. However, there can also be additional charged residues at the active site, which promote substrate recognition and binding <cite>Thomas2013</cite>. Such residues can be found in the N-terminal R*ELREML and VIIGQIH regions. Both highly conserved regions are mainly characterized by hydrophobic amino acids (especially aromatic amino acids) such as leucine, tryptophan and methionine as well as residues with planar polar side chains (especially amino acids with charged side chains) such as arginine, glutamic acid, glutamine (Figure 2). These residues have been suggested to be substrate-binding molecules <cite>Wong2000</cite>.

== Substrate specificities ==
[[Image:PL7SF_Thomasetal2013.JPG|thumb|400px|'''Figure 2. Subfamilies of PL7s''' <cite>Thomas2013</cite>. Unrooted tree with bootstrap values after maximum likelihood analysis. Number refer to Uniprot accession numbers. Red dots indicate enzymes from ''Z. galactanivorans''. Pink triangles indicate enzymes characterized biochemically. Blue squares indicate that the structure of the protein has been solved.]]
Polysaccharide lyase family 7 (PL7) contains five subfamilies (SF) based on their sequence similarities <cite>Lombard2010</cite>, plus a so far uncharacterized sixth subfamily, which consist only of marine representatives of the Flavobacteriaceae (Figure 2) <cite>Thomas2013</cite>. The substrate specificity depends on the source of alginate, i.e. derived from brown seaweed or mucoid bacteria ''Pseudomonas'' spp. and ''Azotobacter vinelandii'', as well as geographical and seasonal parameters. Alginate is a heteropolysaccharide, consisting of β-D-mannuronate (M) and α-L-guluronate (G). These monosaccharides can occur in homogenous and heterogenous blocks. In addition, bacterial alginate is often acetylated at the C2 and / or C3 of mannuronate, thereby shielding the substrate from degradation. Hence, PL7s can be mannuronate ([{{EClink}}4.2.2.3 EC 4.2.2.3]), guluronate ([{{EClink}}4.2.2.11 EC 4.2.2.11]) or mixed link (EC 4.2.2.-) specific lyases. Despite the preference for M- or G-enriched blocks, most PL7 also have a low to moderate activity for the other building block <cite>Thomas2013 Badur2015 Sim2017</cite>. PolyG specific PL7 have been found in the SF3 and SF5 <cite>Thomas2013</cite> with QIH in the second highly conserved region, while polyM specific PL7s are characterized by QVH <cite>Zhu2015 Deng2014</cite>.

== Three-dimensional structures ==
[[Image:EndovsexoPL7_Thomas_et_al_2013.jpg|thumb|400px|'''Figure 3. 3D Structure of endo- and exo-active PL7s''' <cite>Thomas2013</cite>. (A,B) endo AlyA1 and (C, D) exo AlyA5 from ''Zobellia galaactinovorans'' DsijT shown as cartoon (A,C) and surface structure (B,D) with superimposed tetrasaccharide from [{{PDBlink}}2ZAA PDB ID 2ZAA]. The image was conducted in PyMOL <cite>DeLano2002</cite>.]]
PL7 displays a jelly roll fold, which is also found in PL14 as well as in glycoside hydrolases of family [https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_16 GH16]. ''Zobellia galactanivorans'' DsijT possess, among others, two PL7 with different modes of activity <cite>Thomas2013</cite>. AlyA1 is an endo-acting PL7 belonging to SF3 with a wide open cleft harboring the active site (Figure 3A, B), whereas AlyA5 belongs to SF5 and is exo-active which active site is closed by three loops forming a small pocket (Figure 3C, D). The highly conserved 9-amino-acid-block YFKAGVY*Q (where * is a variable residue) at the C-terminus of PL7s (Figure 2) has also been found for an extracellular pectate lyase (PL1, PDB: 1AIR) in ''E. chrysanthemi''. Alginate and pectate/pectin lyases share the β-elimination mechanism, the recognition of substrates of a similar structure, Ca<sup>2+</sup>-binding site and primary sequence similarity, indicating that they probably possess a similar core structural fold probably important to maintain a stable 3D-confirmation <cite>Wong2000</cite>.
Several alginate lyases have been reported to be multimodular enzymes with putative non-catalytic, carbohydrate-binding modules ([https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Families CBMs]). The first biochemically characterized CBM of an endo PL7 was a N-terminal CBM13 from ''Agarivorans'' sp. L11, which increased its substrate binding and therefore catalytic efficiency, influenced substrate specificity, the product profile and thermo stability <cite>Li2015</cite>. Opposite observations regarding catalytic efficiency and substrate specificity were made for an endo PL7 from ''Vibrio splendidus'' OU02 DNA with an N-terminal [https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Family_32 CBM32] linked by a unique alpha-helix linker. The CBM and linker were proposed to serve as a "pivot point" somehow just pushing the product profile towards trisaccharides <cite>Lyu2018</cite>. The PL7 from ''Persicobacter'' sp. CCB-QB2 consists of three domains - a N-terminal [https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Family_16 CBM16] with unknown function, a [https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Family_32 CBM32] and the C-terminal PL7 <cite>Sim2017</cite>. Overall the role of [https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Families CBMs] from different families as part of PL7s still remains unclear. Another common structural feature found in PLs are flexible loops facing above the active site <cite>Xu2018</cite>, which have been demonstrated to influence substrate recognition, binding <cite>Thomas2013 Ogura2008</cite> and potentially specificity <cite>Qin2018</cite>.

== Gene transfer of Alys among different habitats ==
Alginate degrading organisms often posse specified gene clusters for glycan utilization which contain, among other proteins such as transporters, several endo- and exo-acting Alys of different families. These gene clusters are called polysaccharide utilization loci (PULs) for Bacteriodetes <cite>Bjursell2016</cite> or alginolytic operons in case a SusCD pair transporter is missing or replaced by a different transporter system. It has been shown that these gene clusters can be transferred horizontally from one organism to another and thereby even cross different environmental habitats <cite>Hehemann2010 Mathieu2018</cite>. The first alginate utilization system (AUS) was found in ''Zobellia galactinovorans'' which contains two clusters harboring five out of seven Alys (3x PL7). Those operons originated from an ancestral marine Flavobacterium and were independently transferred to marine Proteobacteria and Japanese gut Bacteriodetes by lateral gene transfer (LGT) <cite>Thomas2012</cite>.

A more detailed studied on horizontal gene transfer of PL7 between marine, ecophysiological different Vibrionaceae isolates revealed rapid adaptation of closely related but speciated bacterial populations, resulting into a fine scale of resource partitioning. The exchange of PL7 between marine microbes drove the evolution of polysaccharide degrading pathways, which might have led to three ecotypes – the pioneers, which degrade the polymer into oligomers, the harvester (intermediate of the other two types) and the scavenger, which can only utilize very small oligos created by the pioneers <cite>Hehemann2016</cite>.

== Family Firsts ==
;First catalytic endo-activity: polyM PL7 from ''Photobacterium'' ATCC 433367 <cite>Malissard1993</cite>, polyG PL7 from ''Klebsiella pneumoniae subbsp. aerogenes'' <cite>Baron1994</cite>
;First catalytic exo-activity: AlyA5 from ''Zobellia galactanivorans'' DsijT <cite>Thomas2013</cite>
;First 3-D apo-structure: PA1167 from ''Pseudomonas aeruginosa'' <cite>Yamasaki2004</cite>
;First 3-D holo-structure: A1-II from ''Sphingomons'' sp. A1 <cite>Ogura2007</cite>
;First characterised CBM: AlyL2 containing a N-terminal CBM13 from ''Agarivorans'' sp. L11 <cite>Li2015</cite>

== References ==
<biblio>
#Lombard2010 pmid=20925655
#Lombard2014 pmid=24270786
#Favorov1979 pmid=476128
#Gacesa1986 Gacesa P. (1987) Alginate‐modifying enzymes: A proposed unified mechanism of action for the lyases and epimerases. ''FEBS Letters'', '''212''', 1873-3468. [http://dx.doi.org/10.1016/0014-5793(87)81344-3 DOI:10.1016/0014-5793(87)81344-3]
#Thomas2013 pmid=23782694
#Thomas2012 pmid=22513138
#Hehemann2010 pmid=20376150
#Yamasaki2004 pmid=15136569
#Badur2015 pmid=25556193
#Yamasaki2005 pmid=16081095
#Deng2014 pmid=24050202
#Li2015 pmid=25837818
#Zhu2015 pmid=25831216
#Wong2000 pmid=11018131
#Sim2017 pmid=29057942
#Lyu2018 pmid=29864445
#Bjursell2016 pmid=16968696
#Malissard1993 pmid=8319887
#Baron1994 pmid=8200539
#Osawa2015 pmid=15644208
#Robert2014 pmid=24753421
#DeLano2002 Delano WL. (2002) Pymol: An open-source molecular graphics tool. ''CCP4 Newsletter On Protein Crystallography'', '''40''', 82-92. [http://www.ccp4.ac.uk/newsletters/newsletter40.pdf#page=44 Direct link].
#Preston2000 pmid=11029455
#Jagtap2014 pmid=24795372
#Hehemann2016 pmid=27653556
#Mathieu2018 pmid=29795267
#Ogura2008 pmid=18514736
#Xu2018 pmid=29150496
#Qin2018 pmid=29292806
#Grant1973 Grant, G., Morris, E., Rees, D., Smith, P., and Thom, D.Biological interactions between polysaccharides and diva-lent cations: the egg-box model. FEBS Lett32,195, 1973.
</biblio>

[[Category:Polysaccharide Lyase Families|PL007]]

Polysaccharide Lyase Family 7

2019-08-06T12:22:21Z

2012-07-04T05:19:42Z

Jan-Hendrik Hehemann:

Glycoside Hydrolase Family 117

2012-07-04T05:19:21Z

Jan-Hendrik Hehemann:

2012-05-30T05:27:42Z

Jan-Hendrik Hehemann:

Glycoside Hydrolase Family 16

2012-05-27T06:11:54Z

Jan-Hendrik Hehemann:

{{CuratorApproved}}
* [[Author]]: [[User:JensEklof|Jens Eklöf]] and ^^^Jan-Hendrik Hehemann^^^
* [[Responsible Curator]]: ^^^Harry Brumer^^^
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 16'''
|-
|'''Clan'''
|GH-B
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH16.html
|}
</div>

== Substrate specificities ==
[[Glycoside hydrolases]] of family 16 cleave β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. Some members of this family operating on xyloglucan exhibit predominant ''[[endo]]''-transglycosylase activity <cite>Baumann2007</cite>.
The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC [{{EClink}}2.4.1.207 2.4.1.207]),
keratan-sulfate ''[[endo]]''-1,4-β-galactosidases (EC [{{EClink}}3.2.1.103 3.2.1.103]), ''[[endo]]''-1,3-β-galactanases (EC [{{EClink}}3.2.1.* 3.2.1.-]), ''[[endo]]''-1,3-β-glucanases (EC [{{EClink}}3.2.1.39 3.2.1.39]), ''[[endo]]''-1,3(4)-β-glucanases (EC [{{EClink}}3.2.1.6 3.2.1.6]), lichenases (EC [{{EClink}}3.2.1.73 3.2.1.73]), β-agarases (EC [{{EClink}}3.2.1.81 3.2.1.81]), β-porphyranases (EC [{{EClink}}3.2.1.178 3.2.1.178]) <cite>Hehemann2010</cite>, κ-carrageenases (EC [{{EClink}}3.2.1.83 3.2.1.83]) and xyloglucanases (EC [{{EClink}}3.2.1.151 3.2.1.151]).

Some invertebrate GH16 proteins have lost their catalytic amino acids and are involved in immune response activation through the Toll pathway upon binding of β-1,3 glucan. The role of the GH16 domain in this immune response is still not elucidated <cite>Lee2009</cite>.

<gallery widths=270px perrow=3 caption="Polysaccharides cleaved by GH16 enzymes">

File:AgarIII.png|'''Agar''' <br> β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''

File:Kappa_carrageenan.png|'''κ-carrageenan''' <br> β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''

File:porphyran.png|'''Porphyran''' <br>

File:laminaritetraose.png|'''β-1,3-glucan''' <br> β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''

File:keratan_sulphate.png|'''Keratan sulphate''' <br>

File:beta_13_galactan.png|'''β-1,3-galactan''' <br> β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''

File:MLG_pentaose.png|'''β-1,4/1,3-glucan''' <br> β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,4)- β-D-Glc''p''

File:beta_14_galactan.png|'''β-1,4-galactan''' <br> β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''

File:Xyloglucan.png|'''Xyloglucan''' <br>

</gallery>
== Kinetics and Mechanism ==
Family 16 enzymes are [[retaining]] enzymes, as first shown by NMR <cite>Malet1993</cite> on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus licheniformis''.

== Catalytic Residues ==
The [[catalytic nucleophile]] was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and subsequent peptide identification by ESI-MS and Edman degradation on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus amyloliquefaciens'' <cite>Hoj1992</cite>. This was subsequently verified by azide rescue of the E134A mutant of a ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase resulting in an α-glycosyl azide from the β-glycoside substrate <cite>Viladot1998</cite>. The [[general acid/base]] residue was identified by making the E138A mutant from the ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase and subsequent azide rescue resulting in a β-glycosyl azide product <cite>Viladot1998</cite>. This mechanistic analysis on bacterial mixed-linkage [[endo]]-glucanases has been reviewed in the broader context of GH16 <cite>Planas2000</cite>.

== Three-dimensional structures ==
Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3D structure was a hybrid protein of lichenase M from ''Paenibacillus macerans'' and BglA from ''Bacillus amyloliquefaciens'' ([{{PDBlink}}1byh PDB 1byh]) in 1992 <cite>Keitel1993</cite>.
The first eukaryotic 3D structure was the xyloglucan ''endo''-transglycosylase ''Ptt''XET16-34 from ''Populus tremula×tremuloides'' ([{{PDBlink}}1umz PDB 1umz]) <cite>Johansson2004</cite>. The first archeal 3D structure was a ''[[endo]]''-1,3-β-glucanase Lam16 from ''Pyrococcus furiosus'' ([{{PDBlink}}2vy0 PDB 2vy0]) <cite>Ilari2009</cite>.

== Evolution of GH16 ==
[[Image:TreeGH16new.png|thumb|right|450px|Evolution of family 16 (''click to enlarge'')]]
Family 16 is a member of [[clans|clan]] GH-B together with family 7 with whom they share their β-jellyroll fold. The different specificities of family 16 has been proposed to have evolved from an ancestral β-1,3-glucanase <cite>Barbeyron1998</cite>. The first branching in family 16 lead to the evolution of the κ-carrageenases and the β-agarases and a later branching event lead to the arisal of the lichenases and the XETs <cite>Michel2001</cite> (see figure). This evolutionary scenario was further developed and supported by using a structure based phylogeny approach. In GH16 the active site residues are located in one beta-strand at the center of the substrate binding cleft and encoded within the signature motive EXDXXE or EXDXE. These motives feature two topologies, the beta-bulge motive which is more frequent in GH16 compared to the regular beta-strand, in which one amino acid is deleted. Due to the large expansion of the beta-bulge motive and its appearance in the related GH7 Michel et al. proposed that the ancestral enzyme of both families contained the beta-bulge explaining its wide distribution in GH16. This motive subsequently evolved to become the regular beta-strand that is common in contemporary XETs and lichenases.

== Family firsts ==
; First stereochemistry determination : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR <cite>Malet1993</cite>.
; First [[catalytic nucleophile]] identification : Suggested in ''Bacillus amyloliquefaciens'' 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labelling<cite>Hoj1992</cite>. Later verified in by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First [[general acid/base]] residue identification : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase, first suggested by sequence homology and mutational studies <cite>Juncosa1994</cite>. This was later verified by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First 3-D structure : A hybrid lichenase (''Bacillus amyloliquefaciens'' and ''Paenibacillus macerans'') by X-ray crystallography ([{{PDBlink}}1byh PDB 1byh]) <cite>Keitel1993</cite>.

== Reference list ==
<biblio>
#Johansson2004 pmid=15020748
#Hoj1992 pmid=1360982
#Malet1993 pmid=8280073
#Keitel1993 pmid=8099449
#Juncosa1994 pmid=8182059
#Viladot1998 pmid=9698381
#Ilari2009 pmid=19154353
#Michel2001 pmid=11435116 tree GH16
#Barbeyron1998 pmid=9580981 first GH16 paper
#Baumann2007 pmid=17557806
#Planas2000 pmid=11150614
#Hehemann2010 pmid=20376150
#Kotake2011 pmid=21653698
#Lee2009 pmid=19712587
</biblio>


[[Category:Glycoside Hydrolase Families|GH016]]

Glycoside Hydrolase Family 86

2012-05-27T03:19:32Z

Jan-Hendrik Hehemann:

{{CuratorApproved}}
* [[Author]]: ^^^Mirjam Czjzek^^^
* [[Responsible Curator]]: ^^^Mirjam Czjzek^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH86'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|probably retaining
|-
|'''Active site residues'''
|inferred from clan GH-A as two Glu
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH86.html
|}
</div>


== Substrate specificities ==
[[Glycoside hydrolases]] of family 86 are β-agarases (EC [{{EClink}}3.2.1.81 3.2.1.81]) that cleave β-1,4 glycosidic bonds of agarose. As of November 2010, only three enzymes have been characterized: AgrA from ''Pseudoalteromonas atlantica'', AgaO from ''Microbulbifer thermotolerans JAMB-A94'' and Aga86E from ''Saccharophagus degradans 2-40'' <cite>Belas1989,Ohta2004,Ekborg2006</cite>. AgaO from ''M. thermotolerans'' was reported to be an endo-hydrolytic enzyme, releasing neoagaro-hexaose as main product <cite>Ohta2004</cite>, while the recombinant Aga86E from ''S. degradans'' released only neoagarobiose in an exo-acting manner <cite>Ekborg2006</cite>.

== Kinetics and Mechanism ==
A potential retaining mechanism of this glycoside hydrolase family can only be inferred from analogy to clan [{{CAZyDBlink}}Glycoside-Hydrolases.html GH-A enzymes]. No mechanistic or kinetic analysis demonstrating the stereochemical outcome of the reaction have been reported for this family to date.

== Catalytic Residues ==
Actually, the catalytic residues can only be inferred from analogy to clan GH-A enzymes as two glutamate residues.

== Three-dimensional structures ==
No 3D structure is available to date.

== Family Firsts ==
;Identification of first family member: The first member of this family, AgrA, was identified in ''Pseudoalteromonas atlantica'' <cite>Belas1989</cite>.
;First stereochemistry determination: not determined yet.
;First catalytic nucleophile identification: not determined yet.
;First general acid/base residue identification: not determined yet.
;First 3-D structure: not determined yet.

== References ==
<biblio>
#Belas1989 pmid=2914859
#Ohta2004 pmid=15490156
#Ekborg2006 pmid=16672483

</biblio>

[[Category:Glycoside Hydrolase Families|GH086]]

Glycoside Hydrolase Family 16

2012-05-23T19:56:43Z

Jan-Hendrik Hehemann:

{{CuratorApproved}}
* [[Author]]: [[User:JensEklof|Jens Eklöf]] and ^^^Jan-Hendrik Hehemann^^^
* [[Responsible Curator]]: ^^^Harry Brumer^^^
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 16'''
|-
|'''Clan'''
|GH-B
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH16.html
|}
</div>

== Substrate specificities ==
[[Glycoside hydrolases]] of family 16 enzymes cleave β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. Some members of this family operating on xyloglucan exhibit predominant ''[[endo]]''-transglycosylase activity <cite>Baumann2007</cite>.
The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC [{{EClink}}2.4.1.207 2.4.1.207]),
keratan-sulfate ''[[endo]]''-1,4-β-galactosidases (EC [{{EClink}}3.2.1.103 3.2.1.103]), ''[[endo]]''-1,3-β-galactanases (EC [{{EClink}}3.2.1.* 3.2.1.-]), ''[[endo]]''-1,3-β-glucanases (EC [{{EClink}}3.2.1.39 3.2.1.39]), ''[[endo]]''-1,3(4)-β-glucanases (EC [{{EClink}}3.2.1.6 3.2.1.6]), lichenases (EC [{{EClink}}3.2.1.73 3.2.1.73]), β-agarases (EC [{{EClink}}3.2.1.81 3.2.1.81]), β-porphyranases (EC [{{EClink}}3.2.1.178 3.2.1.178]) <cite>Hehemann2010</cite>, κ-carrageenases (EC [{{EClink}}3.2.1.83 3.2.1.83]) and xyloglucanases (EC [{{EClink}}3.2.1.151 3.2.1.151]).

Some invertebrate GH16 proteins have lost their catalytic amino acids and are involved in immune response activation through the Toll pathway upon binding of β-1,3 glucan. The role of the GH16 domain in this immune response is still not elucidated <cite>Lee2009</cite>.

<gallery widths=270px perrow=3 caption="Polysaccharides cleaved by GH16 enzymes">

File:AgarIII.png|'''Agar''' <br> β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''

File:Kappa_carrageenan.png|'''κ-carrageenan''' <br> β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''

File:porphyran.png|'''Porphyran''' <br>

File:laminaritetraose.png|'''β-1,3-glucan''' <br> β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''

File:keratan_sulphate.png|'''Keratan sulphate''' <br>

File:beta_13_galactan.png|'''β-1,3-galactan''' <br> β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''

File:MLG_pentaose.png|'''β-1,4/1,3-glucan''' <br> β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,4)- β-D-Glc''p''

File:beta_14_galactan.png|'''β-1,4-galactan''' <br> β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''

File:Xyloglucan.png|'''Xyloglucan''' <br>

</gallery>
== Kinetics and Mechanism ==
Family 16 enzymes are [[retaining]] enzymes, as first shown by NMR <cite>Malet1993</cite> on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus licheniformis''.

== Catalytic Residues ==
The [[catalytic nucleophile]] was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and subsequent peptide identification by ESI-MS and Edman degradation on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus amyloliquefaciens'' <cite>Hoj1992</cite>. This was subsequently verified by azide rescue of the E134A mutant of a ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase resulting in an α-glycosyl azide from the β-glycoside substrate <cite>Viladot1998</cite>. The [[general acid/base]] residue was identified by making the E138A mutant from the ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase and subsequent azide rescue resulting in a β-glycosyl azide product <cite>Viladot1998</cite>. This mechanistic analysis on bacterial mixed-linkage [[endo]]-glucanases has been reviewed in the broader context of GH16 <cite>Planas2000</cite>.

== Three-dimensional structures ==
Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3D structure was a hybrid protein of lichenase M from ''Paenibacillus macerans'' and BglA from ''Bacillus amyloliquefaciens'' ([{{PDBlink}}1byh PDB 1byh]) in 1992 <cite>Keitel1993</cite>.
The first eukaryotic 3D structure was the xyloglucan ''endo''-transglycosylase ''Ptt''XET16-34 from ''Populus tremula×tremuloides'' ([{{PDBlink}}1umz PDB 1umz]) <cite>Johansson2004</cite>. The first archeal 3D structure was a ''[[endo]]''-1,3-β-glucanase Lam16 from ''Pyrococcus furiosus'' ([{{PDBlink}}2vy0 PDB 2vy0]) <cite>Ilari2009</cite>.

== Evolution of GH16 ==
[[Image:TreeGH16new.png|thumb|right|450px|Evolution of family 16 (''click to enlarge'')]]
Family 16 is a member of [[clans|clan]] GH-B together with family 7 with whom they share their β-jellyroll fold. The different specificities of family 16 has been proposed to have evolved from an ancestral β-1,3-glucanase <cite>Barbeyron1998</cite>. The first branching in family 16 lead to the evolution of the κ-carrageenases and the β-agarases and a later branching event lead to the arisal of the lichenases and the XETs <cite>Michel2001</cite> (see figure). This evolutionary scenario was further developed and supported by using a structure based phylogeny approach. In GH16 the active site residues are located in one beta-strand at the center of the substrate binding cleft and encoded within the signature motive EXDXXE or EXDXE. These motives feature two topologies, the beta-bulge motive which is more frequent in GH16 compared to the regular beta-strand, in which one amino acid is deleted. Due to the large expansion of the beta-bulge motive and its appearance in the related GH7 Michel et al. proposed that the ancestral enzyme of both families contained the beta-bulge explaining its wide distribution in GH16. This motive subsequently evolved to become the regular beta-strand that is common in contemporary XETs and lichenases.

== Family firsts ==
; First stereochemistry determination : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR <cite>Malet1993</cite>.
; First [[catalytic nucleophile]] identification : Suggested in ''Bacillus amyloliquefaciens'' 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labelling<cite>Hoj1992</cite>. Later verified in by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First [[general acid/base]] residue identification : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase, first suggested by sequence homology and mutational studies <cite>Juncosa1994</cite>. This was later verified by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First 3-D structure : A hybrid lichenase (''Bacillus amyloliquefaciens'' and ''Paenibacillus macerans'') by X-ray crystallography ([{{PDBlink}}1byh PDB 1byh]) <cite>Keitel1993</cite>.

== Reference list ==
<biblio>
#Johansson2004 pmid=15020748
#Hoj1992 pmid=1360982
#Malet1993 pmid=8280073
#Keitel1993 pmid=8099449
#Juncosa1994 pmid=8182059
#Viladot1998 pmid=9698381
#Ilari2009 pmid=19154353
#Michel2001 pmid=11435116 tree GH16
#Barbeyron1998 pmid=9580981 first GH16 paper
#Baumann2007 pmid=17557806
#Planas2000 pmid=11150614
#Hehemann2010 pmid=20376150
#Kotake2011 pmid=21653698
#Lee2009 pmid=19712587
</biblio>


[[Category:Glycoside Hydrolase Families|GH016]]

Glycoside Hydrolase Family 16

2012-03-12T19:36:42Z

Jan-Hendrik Hehemann:

{{CuratorApproved}}
* [[Author]]: [[User:JensEklof|Jens Eklöf]] and ^^^Jan-Hendrik Hehemann^^^
* [[Responsible Curator]]: ^^^Harry Brumer^^^
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 16'''
|-
|'''Clan'''
|GH-B
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH16.html
|}
</div>

== Substrate specificities ==
[[Glycoside hydrolases]] of family 16 enzymes cleave β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. Some members of this family operating on xyloglucan exhibit predominant ''[[endo]]''-transglycosylase activity <cite>Baumann2007</cite>.
The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC [{{EClink}}2.4.1.207 2.4.1.207]),
keratan-sulfate ''[[endo]]''-1,4-β-galactosidases (EC [{{EClink}}3.2.1.103 3.2.1.103]), ''[[endo]]''-1,3-β-galactanases (EC [{{EClink}}3.2.1.* 3.2.1.-]), ''[[endo]]''-1,3-β-glucanases (EC [{{EClink}}3.2.1.39 3.2.1.39]), ''[[endo]]''-1,3(4)-β-glucanases (EC [{{EClink}}3.2.1.6 3.2.1.6]), lichenases (EC [{{EClink}}3.2.1.73 3.2.1.73]), β-agarases (EC [{{EClink}}3.2.1.81 3.2.1.81]), β-porphyranases (EC [{{EClink}}3.2.1.178 3.2.1.178]) <cite>Hehemann2010</cite>, κ-carrageenases (EC [{{EClink}}3.2.1.83 3.2.1.83]) and xyloglucanases (EC [{{EClink}}3.2.1.151 3.2.1.151]).

Some invertebrate GH16 proteins have lost their catalytic amino acids and are involved in immune response activation through the Toll pathway upon binding of β-1,3 glucan. The role of the GH16 domain in this immune response is still not elucidated <cite>Lee2009</cite>.

<gallery widths=270px perrow=3 caption="Polysaccharides cleaved by GH16 enzymes">

File:AgarIII.png|'''Agar''' <br> β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-L-3,6-anhydro-Gal''p''

File:Kappa_carrageenan.png|'''κ-carrageenan''' <br> β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''-1,3-β-D-Gal''p''-(1,4)-α-D-3,6-anhydro-Gal''p''

File:porphyran.png|'''Porphyran''' <br>

File:laminaritetraose.png|'''β-1,3-glucan''' <br> β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,3)- β-D-Glc''p''

File:keratan_sulphate.png|'''Keratan sulphate''' <br>

File:beta_13_galactan.png|'''β-1,3-galactan''' <br> β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''-(1,3)- β-D-Gal''p''

File:MLG_pentaose.png|'''β-1,4/1,3-glucan''' <br> β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,4)- β-D-Glc''p''-(1,3)- β-D-Glc''p''-(1,4)- β-D-Glc''p''

File:beta_14_galactan.png|'''β-1,4-galactan''' <br> β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''-(1,4)- β-D-Gal''p''

File:Xyloglucan.png|'''Xyloglucan''' <br>

</gallery>
== Kinetics and Mechanism ==
Family 16 enzymes are [[retaining]] enzymes, as first shown by NMR <cite>Malet1993</cite> on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus licheniformis''.

== Catalytic Residues ==
The [[catalytic nucleophile]] was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and subsequent peptide identification by ESI-MS and Edman degradation on an ''[[endo]]''-1,3-1,4-β-D-glucan 4-glucanohydrolase from ''Bacillus amyloliquefaciens'' <cite>Hoj1992</cite>. This was subsequently verified by azide rescue of the E134A mutant of a ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase resulting in an α-glycosyl azide from the β-glycoside substrate <cite>Viladot1998</cite>. The [[general acid/base]] residue was identified by making the E138A mutant from the ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase and subsequent azide rescue resulting in a β-glycosyl azide product <cite>Viladot1998</cite>. This mechanistic analysis on bacterial mixed-linkage [[endo]]-glucanases has been reviewed in the broader context of GH16 <cite>Planas2000</cite>.

== Three-dimensional structures ==
Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3D structure was a hybrid protein of lichenase M from ''Paenibacillus macerans'' and BglA from ''Bacillus amyloliquefaciens'' ([{{PDBlink}}1byh PDB 1byh]) in 1992 <cite>Keitel1993</cite>.
The first eukaryotic 3D structure was the xyloglucan ''endo''-transglycosylase ''Ptt''XET16-34 from ''Populus tremula×tremuloides'' ([{{PDBlink}}1umz PDB 1umz]) <cite>Johansson2004</cite>. The first archeal 3D structure was a ''[[endo]]''-1,3-β-glucanase Lam16 from ''Pyrococcus furiosus'' ([{{PDBlink}}2vy0 PDB 2vy0]) <cite>Ilari2009</cite>.

== Evolution of GH16 ==
[[Image:TreeGH16new.png|thumb|right|450px|Evolution of family 16 (''click to enlarge'')]]
Family 16 is a member of [[clans|clan]] GH-B together with family 7 with whom they share their β-jellyroll fold. The different specificities of family 16 has been proposed to have evolved from an ancestral β-1,3-glucanase <cite>Barbeyron1998</cite>. The first branching in family 16 lead to the evolution of the κ-carrageenases and the β-agarases and a later branching event lead to the arisal of the lichenases and the XETs <cite>Michel2001</cite> (see figure).

== Family firsts ==
; First stereochemistry determination : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR <cite>Malet1993</cite>.
; First [[catalytic nucleophile]] identification : Suggested in ''Bacillus amyloliquefaciens'' 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labelling<cite>Hoj1992</cite>. Later verified in by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First [[general acid/base]] residue identification : ''Bacillus licheniformis'' 1,3-1,4-β-D-glucan 4-glucanohydrolase, first suggested by sequence homology and mutational studies <cite>Juncosa1994</cite>. This was later verified by azide rescue of inactivated mutants <cite>Viladot1998</cite>.
; First 3-D structure : A hybrid lichenase (''Bacillus amyloliquefaciens'' and ''Paenibacillus macerans'') by X-ray crystallography ([{{PDBlink}}1byh PDB 1byh]) <cite>Keitel1993</cite>.

== Reference list ==
<biblio>
#Johansson2004 pmid=15020748
#Hoj1992 pmid=1360982
#Malet1993 pmid=8280073
#Keitel1993 pmid=8099449
#Juncosa1994 pmid=8182059
#Viladot1998 pmid=9698381
#Ilari2009 pmid=19154353
#Michel2001 pmid=11435116 tree GH16
#Barbeyron1998 pmid=9580981 first GH16 paper
#Baumann2007 pmid=17557806
#Planas2000 pmid=11150614
#Hehemann2010 pmid=20376150
#Kotake2011 pmid=21653698
#Lee2009 pmid=19712587
</biblio>


[[Category:Glycoside Hydrolase Families|GH016]]

File:TreeGH16new.png

2012-03-12T19:33:52Z

Jan-Hendrik Hehemann:

2012-01-19T02:18:11Z

Jan-Hendrik Hehemann:

[[Image:JH.jpg|200px|right]]
I studied biochemistry and molecular biology at the University of Hamburg (Germany) and graduated in 2005. In 2006 I received an early stage Marie Curie fellowship to start a PhD, with [http://www.cazypedia.org/index.php/User:Mirjam_Czjzek Dr. Mirjam Czjzek] at the [http://www3.sb-roscoff.fr/Station Biologique de Roscoff] (France). During my PhD I worked on glycoside hydrolases, from the marine flavobacterium ''Zobellia galactanivorans'', specific for cell wall polysaccharides from red algae. I focused on a new subfamily in [[GH16]], of which the specificity was unknown, and discovered that they are beta-porphyranases. These enzymes degrade the polysaccharide porphyran from red seaweeds like ''Porphyra'' sp. which is more generally known as Nori and used to make one of my favourite foods, the Maki-Sushi roll. When we searched for other porphyranases in public sequence databases we found them solely in genomes of marine bacteria and not in terrestrial bacteria probably because porphyran is an algal polysaccharide and absent in terrestrial plants. We found however an exception, one porphyranase was encoded in the genome of ''Bacteroides plebeius'', a human gut bacterium that was isolated from a Japanese individual. A subsequent analysis of available gut metagenome datasets revealed that porphyranases are common in gut bacteria from the Japanese people and so far absent in others. We suggested that the consumption of non sterile and fresh food with associated bacteria -“The Sushi Factor”- created contact between bacteria from the ocean and the human gut and led to the horizontal gene transfer of porphyranase genes <cite>Hehemann2010</cite>. This research was widely covered in the press and you can find a great and more palatable article about our paper [http://blogs.discovermagazine.com/notrocketscience/2010/04/07/gut-bacteria-in-japanese-people-borrowed-sushi-digesting-genes-from-ocean-bacteria/ here].

Since March 2010 I am working as a PostDoc with ^^^Alisdair Boraston^^^ at the University of Victoria (Canada) and in August 2010 I received an EMBO Long-Term Fellowship to work on ''B. plebeius'' and how this microbe benefitted through its update from the sea.

In addition to human gut microbes I am very interested in organic matter degradation in the ocean and how marine bacteria degrade algal polysaccharides. This is an exciting and important field of research because the CAZymes used to degrade marine algae are largely unknown, which allows us to make exciting discoveries.
== Reference list ==
<biblio>
#Hehemann2010 pmid=20376150
</biblio>

[[Category:Contributors|Hehemann, Jan-Hendrik]]