Carbohydrate Binding Module Family 79

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

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CAZy DB link

http://www.cazy.org/CBM79.html

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 [1]. Two CBM79s (CBM79-1_RfGH9 and CBM79-2_RfGH9) were identified in an enzyme that contains a catalytic module derived from GH9 with endo-β1,4-glucanases activity. Both CBM79s bind to a range of β-1,4- and mixed linked β-1,3-1,4-glucans [2]. The ligand binding of CBM79-1_RfGH9 was quantified by isothermal titration calorimetry and semi-quantitatively using microarrays [2]. CBM79-1_RfGH9 binds barley β-glucan and hydroxyethylcellulose (HEC) with similar affinities. The small increase in KA from cellotetraose to 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. CBM79-1_RfGH9 binds regenerated (noncrystalline) insoluble cellulose (RC) with a K_A of 4.8 x 10⁴ M^-1 .

Structural Features

Figure 1. Crystal structure of CBM79-1_RfGH9. (PDB ID 4V1L)(PDB ID 4V1K). The aromatic residues that contribute to ligand recognition are shown.

The structure of CBM79-1_RfGH9 was solved using single-wavelength anomalous diffraction (SAD) methods and selenomethionyl protein to a resolution of 1.8 Å. CBM79-1_RfGH9 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. Tyr563, Trp564, Tyr597, Trp606 and Trp607 in CBM79-1RfGH9 form a twisted hydrophobic platform. In CBM79-1_RfGH9, the concave surface forms an unusually solvent exposed cleft or planar surface, with loops connecting β-strands 1 and 2 and β-strands 4 and 5 strongly contributing to the curved topology of β-sheet 2. Two tryptophan residues (Trp564 and Trp606) play a key role in ligand recognition, adopting a planar orientation in CBM79-1_RfGH9.

Functionalities

The specificity of CBM79-1_RfGH9 for β-glucans is consistent with endo-β1,4-glucanase activity of catalytic module GH9. Mutagenesis experiments [2] confirmed the importance of the aromatic residues in ligand recognition. Alanine substitution of Trp606 in CBM79-1_RfGH9, which is conserved in the CBM family, resulted in complete loss of binding to all ligands. The mutants W564A and W607A retained affinity for barley β-glucan but did not bind xyloglucan. The planar topology of the binding cleft of CBM79-1_RfGH9 indicated that this protein may interact with components of insoluble cellulose. ITC and pull down assays showed that CBM79-1_RfGH9 binds RC [2]. Alanine substitution of W564 and W606 resulted in loss of binding to RC. The importance of conformation of conserved aromatic residues on CBM specificity is evident in family 2 CBMs that bind to cellulose or xylan [3].

Family Firsts

First Identified

CBM79 from the Ruminococcus flavefaciens CBM79_1_RfGH9 and CBM79_2_RfGH9 [2].

First Structural Characterization

The first available crystal structure and the first complex structure of a CBM79 is from CBM79_1_RfGH9 [2].

References

<biblio>

1. RinconMT2010 pmid=20814577
2. VendittoI2016 pmid=27298375
3. SimpsonPJ2000 pmid=10973978