Carbohydrate Binding Module Family 78

Ligand specificities

CBM78 is a family identified in the Ruminococcus flavefaciens cellulosome, a ruminal cellulolytic bacterium (1). CBM78 is a component of an enzyme that contains a catalytic module derived from GH5_4 and displays specificity for β-1,4- and mixed linked β-1,3-1,4-glucans. CBM78 also binds galactomannan and contains a GH26 “β1,4-mannanase” catalytic module (2). CBM78_GH5 displays highest affinity for xyloglucan. The similar affinity of CBM78_GH5 for cellohexaose and cellopentaose suggests five dominant sugar binding sites. The higher affinity of CBM78GH5 for XXXG than Cellotetraose, suggests a recognition of the xylose side chains. No binding to regenerated (noncrystalline) insoluble cellulose (RC) is detected.

Structural Features

The structure of CBM78_RfGH5 was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 2.0 Å. CBM78_RfGH5 has a β-sandwich fold and contains two β-sheets, 1 and 2, respectively (Figure 1) (2). β-sheet 2 forms a cleft in which aromatic residues are a dominant feature. Trp496, Trp554, Tyr555, and Phe479 are aligned along the cleft. This hydrophobic region is the glucan binding site in CBM78_RfGH5 (2).

Functionalities

CBM78 plays an enzyme-targeting role that is specific to Ruminococcus and a contribution to enzyme function in a highly complex scaffolding. CBM78 that binds β-glucans is component of enzyme that contains catalytic modules derived from GH5_4 with endo-β1,4-glucanases activity. CBM78 is also component of enzyme that contains catalytic modules derived from GH26 with β1,4-mannanase activity. Mutagenesis experiments confirmed the importance of the aromatic residues in ligand recognition of CBM78RfGH5. Alanine substitution of Trp496 or Trp554 in CBM78_RfGH5, which are conserved in the CBM family, resulted in complete loss of binding to all ligands. The mutants F479A and Y555A bound to xyloglucan, but not to barley β-glucan or HEC.The variant Q552A recognized xyloglucan and barley β-glucan, but not HEC. No binding to regenerated (noncrystalline) insoluble cellulose is detected and it is explained by the narrow binding cleft of CBM78_RfGH5.The mutagenesis data show that different residues play distinct roles in ligand recognition, explaining why this CBM can bind to a range of β-glucans.

Family Firsts

First Identified

CBM78 from the Ruminococcus flavefaciens CBM78_RfGH5 and CBM78_RfGH26 .

First Structural Characterization

The first available crystal structure and the first complex structure of a CBM78 is from CBM78_RfGH5.

References

1. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |

2. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.

3. Boraston AB, Bolam DN, Gilbert HJ, and Davies GJ. (2004). Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004;382(Pt 3):769-81. DOI:10.1042/BJ20040892 |
4. Hashimoto H (2006). Recent structural studies of carbohydrate-binding modules. Cell Mol Life Sci. 2006;63(24):2954-67. DOI:10.1007/s00018-006-6195-3 |
5. Shoseyov O, Shani Z, and Levy I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev. 2006;70(2):283-95. DOI:10.1128/MMBR.00028-05 |
6. Guillén D, Sánchez S, and Rodríguez-Sanoja R. (2010). Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol. 2010;85(5):1241-9. DOI:10.1007/s00253-009-2331-y |
7. Armenta S, Moreno-Mendieta S, Sánchez-Cuapio Z, Sánchez S, and Rodríguez-Sanoja R. (2017). Advances in molecular engineering of carbohydrate-binding modules. Proteins. 2017;85(9):1602-1617. DOI:10.1002/prot.25327 |