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Carbohydrate Binding Module Family 79
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Ruminococcus flavefaciens is an anaerobic, cellulolytic bacterium that plays an important role in the ruminal digestion of plant cell walls . 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 . Both CBM79s bind to a range of β-1,4- and mixed-linked β-1,3-1,4-glucans . The ligand binding of CBM79-1RfGH9 was quantified by Isothermal Titration Calorimetry (ITC) and semi-quantified using Microarrays . CBM79-1RfGH9 binds barley β-glucan and hydroxyethylcellulose (HEC) with similar affinities of 104 M-1 . 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 . The binding to xyloglucan is weaker than the other β-glucans, indicating that the protein cannot recognize the xylose side chains . CBM79-1RfGH9 binds regenerated (non-crystalline) insoluble cellulose (RC) with a KA of 4.8 x 104 M-1 . Mutagenesis experiments confirmed the importance of the aromatic residues in ligand recognition . Alanine substitution of Trp606 in CBM79-1RfGH9, which is conserved in the CBM family, resulted in complete loss of binding to all ligands . The mutants Trp564A and Trp607A retained affinity for barley β-glucan but did not bind xyloglucan . Alanine substitution of Trp564 and Trp606 resulted in loss of binding to RC . The importance of conformation of conserved aromatic residues on CBM specificity is evident in CBM2 members that bind to cellulose or xylan . CBM79 binding to non-crystalline polysaccharides indicates that CBM79-1RfGH9 is a type B CBM .
The structure of CBM79-1RfGH9 was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 1.8 Å . 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) . β-sheet 2 forms a cleft in which aromatic residues are a dominant feature . 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 . Two tryptophan residues (Trp564 and Trp606) play a key role in ligand recognition adopting a planar orientation in CBM79-1RfGH9 .
CBM79 fulfills an enzyme-targeting role that is specific to Ruminococcus . 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 . 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 . 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 .
- First Identified
- CBM79 from the Ruminococcus flavefaciens CBM79_1RfGH9 and CBM79_2RfGH9 were the first CBM79 members characterized .
- First Structural Characterization
- The first available crystal structure and the first complex structure of a CBM79 is from CBM79_1RfGH9 .
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- 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 |
- 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 |