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Difference between revisions of "Carbohydrate Binding Module Family 79"

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== Ligand specificities ==
 
== Ligand specificities ==
CBM79 is a family identified in the ''Ruminococcus flavefaciens'' cellulosome, an anaerobic, cellulolytic bacterium that plays an important role in the ruminal digestion of plant cell walls <cite>RinconMT2010</cite>. Two CBM79s (CBM79-1<sub>RfGH9</sub> and CBM79-2<sub>RfGH9</sub>)  were identified in an enzyme that contains a catalytic module derived from [[GH9]] with endo-β-1,4-glucanase activity <cite>VendittoI2016</cite>. Both CBM79s bind to a range of β-1,4- and mixed linked β-1,3-1,4-glucans <cite>VendittoI2016</cite>. The ligand binding of CBM79-1<sub>RfGH9</sub> was quantified by Isothermal Titration Calorimetry (ITC) and  semi-quantified using Microarrays <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> binds barley β-glucan and hydroxyethylcellulose (HEC) with similar affinities of 10<sup>4</sup> M<sup>-1</sup> <cite>VendittoI2016</cite>. The small increase in ''K''<sub>A</sub> from cellotetraose to cellohexaose  
+
''Ruminococcus flavefaciens'' is an anaerobic, cellulolytic bacterium that plays an important role in the ruminal digestion of plant cell walls <cite>RinconMT2010</cite>. CBM79 is a family identified in the ''Ruminococcus flavefaciens'' cellulosome.  Two CBM79s (CBM79-1<sub>RfGH9</sub> and CBM79-2<sub>RfGH9</sub>)  were identified in an enzyme that contains a [[GH9]] catalytic module with endo-β-1,4-glucanase activity <cite>VendittoI2016</cite>. Both CBM79s bind to a range of β-1,4- and mixed-linked β-1,3-1,4-glucans <cite>VendittoI2016</cite>. The ligand binding of CBM79-1<sub>RfGH9</sub> was quantified by Isothermal Titration Calorimetry (ITC) and  semi-quantified using Microarrays <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> binds barley β-glucan and hydroxyethylcellulose (HEC) with similar affinities of 10<sup>4</sup> M<sup>-1</sup> <cite>VendittoI2016</cite>. The small increase in ''K''<sub>A</sub> from cellotetraose to cellohexaose  
(KA 4.2 x 10<sup>3</sup> M<sup>-1</sup> for cellotetraose, KA 7.0 x 10<sup>3</sup> M<sup>-1</sup> for cellopentaose and KA 4.9 x 10<sup>3</sup> M<sup>-1</sup> for cellohexaose) suggests that ligand recognition is dominated by four sugar binding sites <cite>VendittoI2016</cite>. The binding to xyloglucan is weaker than the other β-glucans, indicating that the protein cannot recognize the xylose side chains <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> binds regenerated (noncrystalline) insoluble cellulose (RC) with a  K<sub>A</sub> of 4.8 x 10<sup>4</sup> M<sup>-1</sup> <cite>VendittoI2016</cite>. The ligand binding site located at the concave surface of the protein forms an unusually solvent exposed cleft or planar surface for a type B glucan binding CBM. It is not a narrow canyon-like structure  as displayed by [[CBM78]], anyway it indicates that CBM79-1<sub>RfGH9</sub> is a [[Carbohydrate-binding_modules#Types|type B]] CBM <cite>VendittoI2016</cite>.
+
(''K''<sub>A</sub> 4.2 x 10<sup>3</sup> M<sup>-1</sup> for cellotetraose, ''K''<sub>A</sub> 7.0 x 10<sup>3</sup> M<sup>-1</sup> for cellopentaose and ''K''<sub>A</sub> 4.9 x 10<sup>3</sup> M<sup>-1</sup> for cellohexaose) suggests that ligand recognition is dominated by four sugar binding sites <cite>VendittoI2016</cite>. The binding to xyloglucan is weaker than the other β-glucans, indicating that the protein cannot recognize the xylose side chains <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> binds regenerated (non-crystalline) insoluble cellulose (RC) with a  ''K''<sub>A</sub> of 4.8 x 10<sup>4</sup> M<sup>-1</sup> <cite>VendittoI2016</cite>. Mutagenesis experiments confirmed the importance of the aromatic residues in ligand recognition <cite>VendittoI2016</cite>. Alanine substitution of Trp606 in CBM79-1<sub>RfGH9</sub>, which is conserved in the CBM family, resulted in complete loss of binding to all ligands <cite>VendittoI2016</cite>. The mutants Trp564A and Trp607A retained affinity for barley β-glucan but did not bind xyloglucan <cite>VendittoI2016</cite>. Alanine substitution of Trp564 and Trp606 resulted in loss of binding to RC <cite>VendittoI2016</cite>. The importance of conformation of conserved aromatic residues on CBM specificity is evident in [[CBM2]] members that bind to cellulose or xylan <cite>SimpsonPJ2000</cite>. CBM79 binding to non-crystalline polysaccharides indicates that CBM79-1<sub>RfGH9</sub> is a [[Carbohydrate-binding_modules#Types|type B]] CBM <cite>VendittoI2016</cite>.
Mutagenesis experiments confirmed the importance of the aromatic residues in ligand recognition <cite>VendittoI2016</cite>. Alanine substitution of Trp606 in CBM79-1<sub>RfGH9</sub>, which is conserved in the CBM family, resulted in complete loss of binding to all ligands <cite>VendittoI2016</cite>. The mutants W564A and W607A retained affinity for barley β-glucan but did not bind xyloglucan <cite>VendittoI2016</cite>. Alanine substitution of W564 and W606 resulted in loss of binding to RC <cite>VendittoI2016</cite>. The importance of conformation of conserved aromatic residues on CBM specificity is evident in family 2 CBMs that bind to cellulose or xylan <cite>SimpsonPJ2000</cite>.  
 
  
 
== Structural Features ==
 
== Structural Features ==
[[File:CBM79-1.jpg|thumb|300px|right|'''Figure 1.'''  Crystal structure of CBM79-1<sub>RfGH9</sub>. ([{{PDBlink}}4V1L PDB ID 4V1L])([{{PDBlink}}4V1K PDB ID 4V1K]). The aromatic residues that contribute to ligand recognition are shown.]]  
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[[File:CBM79-1.jpg|thumb|300px|right|'''Figure 1.'''  Crystal structure of CBM79-1<sub>RfGH9</sub>. ([{{PDBlink}}4V1L PDB ID 4V1L], [{{PDBlink}}4V1K PDB ID 4V1K]). The aromatic residues that contribute to ligand recognition are shown.]]  
The structure of CBM79-1<sub>RfGH9</sub>  was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 1.8 Å <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> displays a beta-sandwich fold in which the 12 antiparallel β-strands are organized in two β-sheets 1 and 2 (Figure 1) <cite>VendittoI2016</cite>. β-sheet 2 forms a cleft in which aromatic residues are a dominant feature <cite>VendittoI2016</cite>. Tyr563, Trp564, Tyr597, Trp606 and Trp607 in CBM79-1<sub>RfGH9</sub> form a twisted hydrophobic platform <cite>VendittoI2016</cite>. Two tryptophan residues (Trp564 and Trp606) play a key role in ligand recognition adopting a planar orientation in CBM79-1<sub>RfGH9</sub> <cite>VendittoI2016</cite>.
+
The structure of CBM79-1<sub>RfGH9</sub>  was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 1.8 Å <cite>VendittoI2016</cite>. CBM79-1<sub>RfGH9</sub> displays a beta-sandwich fold in which the 12 antiparallel β-strands are organized in two β-sheets 1 and 2 (Figure 1) <cite>VendittoI2016</cite>. β-sheet 2 forms a cleft in which aromatic residues are a dominant feature <cite>VendittoI2016</cite>. The ligand binding site located at the concave surface of the protein forms an unusual solvent exposed cleft/planar surface for a [[Carbohydrate-binding_modules#Types|type B]]  β-glucan binding CBM. It is not a narrow canyon-like structure  as displayed by [[CBM78]]. Tyr563, Trp564, Tyr597, Trp606 and Trp607 in CBM79-1<sub>RfGH9</sub> form a twisted hydrophobic platform within the cleft <cite>VendittoI2016</cite>. Two tryptophan residues (Trp564 and Trp606) play a key role in ligand recognition adopting a planar orientation in CBM79-1<sub>RfGH9</sub> <cite>VendittoI2016</cite>.
  
 
== Functionalities ==  
 
== Functionalities ==  
CBM79 fulfills an enzyme-targeting role that is specific to ''Ruminococcus'' <cite>Bensoussan2017</cite>. ''R. flavefaciens'' forms a multi-enzyme cellulosome complex that plays an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides <cite>BergMiller2009</cite>. CBMs generally display specificities consistent with the activity of the appended enzyme. ''R. flavefaciens'' CBM79-1<sub>RfGH9</sub> and CBM79-2<sub>RfGH9</sub> are components of an enzyme that contains a catalytic module derived from [[GH9]] <cite>VendittoI2016</cite>. The specificity of CBM79-1<sub>RfGH9</sub> for β-glucans is consistent with endo-β1,4-glucanase activity of the [[GH9]] catalytic module <cite>VendittoI2016</cite>.  
+
CBM79 fulfills an enzyme-targeting role that is specific to ''Ruminococcus'' <cite>Bensoussan2017</cite>. ''R. flavefaciens'' forms a multi-enzyme cellulosome complex that plays an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides <cite>BergMiller2009</cite>. CBMs generally display specificities consistent with the activity of the appended enzyme. ''R. flavefaciens'' CBM79-1<sub>RfGH9</sub> and CBM79-2<sub>RfGH9</sub> are components of an enzyme that contains a [[GH9]] catalytic module <cite>VendittoI2016</cite>. The specificity of CBM79-1<sub>RfGH9</sub> for β-glucans is consistent with targeting the endo-β1,4-glucanase activity of the [[GH9]] catalytic module to its substrate <cite>VendittoI2016</cite>.  
  
 
== Family Firsts ==
 
== Family Firsts ==
;First Identified: CBM79 from the ''Ruminococcus flavefaciens'' CBM79_1<sub>RfGH9</sub> and CBM79_2<sub>RfGH9</sub> <cite>VendittoI2016</cite>.
+
;First Identified: CBM79 from the ''Ruminococcus flavefaciens'' CBM79_1<sub>RfGH9</sub> and CBM79_2<sub>RfGH9</sub> were the first CBM79 members characterized <cite>VendittoI2016</cite>.
  
 
;First Structural Characterization: The first available crystal structure and the first complex structure of a CBM79 is from CBM79_1<sub>RfGH9</sub> <cite>VendittoI2016</cite>.
 
;First Structural Characterization: The first available crystal structure and the first complex structure of a CBM79 is from CBM79_1<sub>RfGH9</sub> <cite>VendittoI2016</cite>.

Latest revision as of 13:19, 18 December 2021

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Ligand specificities

Ruminococcus flavefaciens is an anaerobic, cellulolytic bacterium that plays an important role in the ruminal digestion of plant cell walls [1]. CBM79 is a family identified in the Ruminococcus flavefaciens cellulosome. Two CBM79s (CBM79-1RfGH9 and CBM79-2RfGH9) were identified in an enzyme that contains a GH9 catalytic module with endo-β-1,4-glucanase activity [2]. Both CBM79s bind to a range of β-1,4- and mixed-linked β-1,3-1,4-glucans [2]. The ligand binding of CBM79-1RfGH9 was quantified by Isothermal Titration Calorimetry (ITC) and semi-quantified using Microarrays [2]. CBM79-1RfGH9 binds barley β-glucan and hydroxyethylcellulose (HEC) with similar affinities of 104 M-1 [2]. The small increase in KA from cellotetraose to cellohexaose (KA 4.2 x 103 M-1 for cellotetraose, KA 7.0 x 103 M-1 for cellopentaose and KA 4.9 x 103 M-1 for cellohexaose) suggests that ligand recognition is dominated by four sugar binding sites [2]. The binding to xyloglucan is weaker than the other β-glucans, indicating that the protein cannot recognize the xylose side chains [2]. CBM79-1RfGH9 binds regenerated (non-crystalline) insoluble cellulose (RC) with a KA of 4.8 x 104 M-1 [2]. Mutagenesis experiments confirmed the importance of the aromatic residues in ligand recognition [2]. Alanine substitution of Trp606 in CBM79-1RfGH9, which is conserved in the CBM family, resulted in complete loss of binding to all ligands [2]. The mutants Trp564A and Trp607A retained affinity for barley β-glucan but did not bind xyloglucan [2]. Alanine substitution of Trp564 and Trp606 resulted in loss of binding to RC [2]. The importance of conformation of conserved aromatic residues on CBM specificity is evident in CBM2 members that bind to cellulose or xylan [3]. CBM79 binding to non-crystalline polysaccharides indicates that CBM79-1RfGH9 is a type B CBM [2].

Structural Features

Figure 1. Crystal structure of CBM79-1RfGH9. (PDB ID 4V1L, PDB ID 4V1K). The aromatic residues that contribute to ligand recognition are shown.

The structure of CBM79-1RfGH9 was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 1.8 Å [2]. CBM79-1RfGH9 displays a beta-sandwich fold in which the 12 antiparallel β-strands are organized in two β-sheets 1 and 2 (Figure 1) [2]. β-sheet 2 forms a cleft in which aromatic residues are a dominant feature [2]. The ligand binding site located at the concave surface of the protein forms an unusual solvent exposed cleft/planar surface for a type B β-glucan binding CBM. It is not a narrow canyon-like structure as displayed by CBM78. Tyr563, Trp564, Tyr597, Trp606 and Trp607 in CBM79-1RfGH9 form a twisted hydrophobic platform within the cleft [2]. Two tryptophan residues (Trp564 and Trp606) play a key role in ligand recognition adopting a planar orientation in CBM79-1RfGH9 [2].

Functionalities

CBM79 fulfills an enzyme-targeting role that is specific to Ruminococcus [4]. R. flavefaciens forms a multi-enzyme cellulosome complex that plays an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides [5]. CBMs generally display specificities consistent with the activity of the appended enzyme. R. flavefaciens CBM79-1RfGH9 and CBM79-2RfGH9 are components of an enzyme that contains a GH9 catalytic module [2]. The specificity of CBM79-1RfGH9 for β-glucans is consistent with targeting the endo-β1,4-glucanase activity of the GH9 catalytic module to its substrate [2].

Family Firsts

First Identified
CBM79 from the Ruminococcus flavefaciens CBM79_1RfGH9 and CBM79_2RfGH9 were the first CBM79 members characterized [2].
First Structural Characterization
The first available crystal structure and the first complex structure of a CBM79 is from CBM79_1RfGH9 [2].

References

  1. Rincon MT, Dassa B, Flint HJ, Travis AJ, Jindou S, Borovok I, Lamed R, Bayer EA, Henrissat B, Coutinho PM, Antonopoulos DA, Berg Miller ME, and White BA. (2010). Abundance and diversity of dockerin-containing proteins in the fiber-degrading rumen bacterium, Ruminococcus flavefaciens FD-1. PLoS One. 2010;5(8):e12476. DOI:10.1371/journal.pone.0012476 | PubMed ID:20814577 [RinconMT2010]
  2. Venditto I, Luis AS, Rydahl M, Schückel J, Fernandes VO, Vidal-Melgosa S, Bule P, Goyal A, Pires VM, Dourado CG, Ferreira LM, Coutinho PM, Henrissat B, Knox JP, Baslé A, Najmudin S, Gilbert HJ, Willats WG, and Fontes CM. (2016). Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition. Proc Natl Acad Sci U S A. 2016;113(26):7136-41. DOI:10.1073/pnas.1601558113 | PubMed ID:27298375 [VendittoI2016]
  3. Simpson PJ, Xie H, Bolam DN, Gilbert HJ, and Williamson MP. (2000). The structural basis for the ligand specificity of family 2 carbohydrate-binding modules. J Biol Chem. 2000;275(52):41137-42. DOI:10.1074/jbc.M006948200 | PubMed ID:10973978 [SimpsonPJ2000]
  4. Bensoussan L, Moraïs S, Dassa B, Friedman N, Henrissat B, Lombard V, Bayer EA, and Mizrahi I. (2017). Broad phylogeny and functionality of cellulosomal components in the bovine rumen microbiome. Environ Microbiol. 2017;19(1):185-197. DOI:10.1111/1462-2920.13561 | PubMed ID:27712009 [Bensoussan2017]
  5. Berg Miller ME, Antonopoulos DA, Rincon MT, Band M, Bari A, Akraiko T, Hernandez A, Thimmapuram J, Henrissat B, Coutinho PM, Borovok I, Jindou S, Lamed R, Flint HJ, Bayer EA, and White BA. (2009). Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1. PLoS One. 2009;4(8):e6650. DOI:10.1371/journal.pone.0006650 | PubMed ID:19680555 [BergMiller2009]

All Medline abstracts: PubMed