Carbohydrate Binding Module Family 78

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• Author: ^^^Immacolata Venditto^^^
• Responsible Curator: ^^^Harry Gilbert^^^

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CAZy DB link

https://www.cazy.org/CBM78.html

Ligand specificities

CBM78 is a family identified in the ruminal cellulolytic bacterium Ruminococcus flavefaciens [1] cellulosome (a lignocellulose degrading multienzyme complex; see [2] for a review of cellulosomes). The only characterized CBM78 modules are CBM78_RfGH5 and CBM78_RfGH26, which are components of enzymes containing catalytic modules derived from GH5_4 and GH26, respectively. While the ligand binding of CBM78_RfGH5 was quantified by isothermal titration calorimetry, the specificity of CBM78_RfGH26 was determined semi-quantitatively using microarrays [3]. Both CBM78 modules bind to β-1,4- and mixed linked β-1,3-1,4-glucans, however, CBM78_RfGH26, also binds galactomannan and contains a GH26 "β1,4-mannanase" catalytic module [3]. CBM78_RfGH5 displays a higher affinity for xyloglucan relative to cellulose ligands. The similar affinity of CBM78_RfGH5 for cellohexaose and cellopentaose suggests five dominant sugar binding sites. The higher affinity of CBM78_RfGH5 for the xyloglucan derived oligosaccharide XXXG (where X is a backbone glucose decorated with xylose and G is an unsubstituted glucose) than cellotetraose suggests recognition of the xylose side chains. No binding to regenerated (noncrystalline) insoluble cellulose (RC) was detected. Isothermal titration calorimetry showed that ligand binding was enthalpically driven and coverage of polysaccharides at saturation indicated an endo-mode of binding. CBM78 is thus a type B CBM family.

Structural Features

Figure 1. Crystal structure of CBM78_RfGH5. (PDB ID 4V17). The aromatic residues that contribute to ligand recognition are shown.

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) [3]. β-sheet 2 forms a cleft in which aromatic residues are a dominant feature, typical of many type B CBM families (see [4] for review). Trp496, Trp554, Tyr555, and Phe479 are aligned along the cleft. This hydrophobic region is the glucan binding site in CBM78_RfGH5 [3].

Functionalities

CBM78 modules play an enzyme-targeting role that is specific to the Ruminococcus, which has a particularly complex repertoire of scaffoldins (cellulosome assembly proteins) [5]. The specificity of CBM78_RfGH5 for β-glucans is consistent with the endo-β1,4-glucanase activity of the cognate GH5_4 catalytic module [6]. Similarly, the capacity of CBM78_RfGH5 to bind β-mannan is consistent with the appended GH26 catalytic module, a family in which the characterized enzymes are predominantly β1,4-mannanases (e.g. [7, 8] and see [9, 10] for reviews). Mutagenesis experiments [3] confirmed the importance of the aromatic residues in ligand recognition of CBM78_RfGH5. 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 hydroxyethylcellulose (HEC). The variant Q552A recognized xyloglucan and barley β-glucan, but not HEC. No binding to regenerated (noncrystalline) insoluble cellulose was detected consistent with the narrow binding cleft of CBM78_RfGH5 [3].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 [3].

First Structural Characterization

The first available crystal structure and the first complex structure of a CBM78 is from CBM78_RfGH5 [3].

References

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7. Hogg D, Pell G, Dupree P, Goubet F, Martín-Orúe SM, Armand S, and Gilbert HJ. (2003). The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation. Biochem J. 2003;371(Pt 3):1027-43. DOI:10.1042/BJ20021860 |