Carbohydrate Binding Module Family 5

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• Author: ^^^Manjeet Kaur^^^
• Responsible Curator: ^^^Appa Rao Podile^^^

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CAZy DB link

http://www.cazy.org/CBM05.html

Ligand specificities

The family 5 carbohydrate binding modules (CBM5) are of approximately 60 residues. These occur as accessory domains in chitinases [1-4], endoglucanases [5] and lytic polysaccharide mono-oxygenases (LPMOs) [6-8]. CBM5 are often reported to have affinity to both cellulose and chitin [1-9]. Deletion of CBM5 from chitinases or LPMOs has shown considerable reduction in chitin binding. The Kd and Bmax values for CBM5 of an LPMO from Cellvibrio japonicas, CjLPMO10A for α-chitin, were 5.3 µM and 4.8 µmol/g α-chitin, respectively [6]. The Kd values of CBM5 from Bacillus thuringiensis, BtCBM5 for α- and β-chitin were in the order of 0.6-0.7 µM, whereas the Bmax value was ~1.9 µmol/g for both α- and β-chitin [8].

Structural Features

The structures of CBM5 domains have been elucidated for an endoglucanase, CBDEGZ from Erwinia chrysanthemi (EcEGZCBM5) and two chitinases ChBDChiB from Serratia marcescens (SmChiBCBM5) and ChBDChiC from Streptomyces griseus HUT6037 (SgChiCCBM5) [1,2,5].

The three structures revealed that CBM5 is composed of five β-strands (β1-5). The β1, β2 and β3 forms the principle structure and the additional short β-strands (β4 and β5) form an antiparallel β-sheet which is independent of the main strand (Figure 1). Brun et al., 1997 reported that EcEGZCBM5 resembles a ski-boot or L-shaped structure composed of only β-sheets. Helix structures have not been found in CBM5 modules.

There are a few differences in CBM5 modules of endo-glucanases (EcEGZCBM5) and chitinases (SmChiBCBM5 and SgChiCCBM5). The EcEGZCBM5 possesses a conserved disulfide bond between Cys4 and Cys61. These disulfide bonds have not been reported in SmChiBCBM5 and SgChiCCBM5.

CBM5 modules possess surface exposed aromatic residues which interact with polysaccharides most probably through hydrophobic interactions. The EcEGZCBM5 possesses three exposed aromatic residues Trp18, Trp43 and Tyr44. Trp18 is present on an extra loop and is linearly aligned to Trp43 and Tyr44 and extends the substrate binding site [4]. The three residues are essential for complete binding of EcEGZCBM5. Polar residues like Asp17 are present on cellulose binding face and form H-bonds to stabilize the appropriate orientation of cellulose binding residues. Polar residues also form H-bonds with oxygen atom and/or OH-groups of glucose subunits of cellulose, thus have been proposed to play a role in cellulose-disruption [5]. Mutation of Asp17 resulted in decreased binding towards cellulose [9].

SmChiBCBM5 and SgChiCCBM5 have only two surface exposed aromatic residues, Trp479 and Trp481 in SmChiBCBM5 and Trp59 and Trp60 in SgChiCCBM5; whose structural homologues in EcEGZCBM5 are Trp43 and Tyr44 [2]. The two exposed aromatic residues are sufficient for binding in SmChiBCBM5 and SgChiCCBM5. These residues interact extensively and play a vital role in increasing the proximity of substrate through hydrophobic interactions. It has been proposed that either of the two exposed residues should be a tryptophan residue. The Tyr-Tyr pair has not been found in the family [1].

In SgChiCCBM5, six residues (Trp36, Val 48, Tyr 50, Tyr55, Pro66 and Trp72) participate in forming a hydrophobic core in the domain centre. The side chain of Pro66 is internally buried while the remaining 5 residues form the hydrophobic socket [1]. Only two surface exposed aromatic residues (Trp59 and Trp60) are involved in carbohydrate binding which are positioned on a loop between the sheets β2 and β5. When protein-substrate interactions were studied between SgChiCCBM5 and tri-N-acetyl-chitotriose, it was found that the ligand binding was facilitated by two stacking interactions (Trp59-NAG-1 and Trp-NAG3) and two H-bonds (Trp60-N and NAG2-O7 and Trp56-NE1 and NAG2-O6) [1].

Functionalities

Multi-modular enzymes like endo-glucanases, chitinases and LPMOs possess CBM5 modules as accessory domains appended to their catalytic domain, either directly or with the help of linkers like FnIII domains [1-8]. The CBM5 domains are responsible for increased affinity of these enzymes towards crystalline cellulose or chitin. Their presence also increases the efficiency of enzymes to bind to substrates in a broader pH range [3, 8]. Deletion of CBM5 domain resulted in reduction or complete loss of binding in several instances [4]. Deletion of C-terminal FnIII and CBM5 domains from BliChi resulted in 5-fold reduction of hydrolytic activity on β-chitin. Also the mutant was unable to degrade α-chitin [4]. Accessory domains have thus been suggested to play an important role in hydrolysis by moving the enzymes in close proximity of substrates. Presence of CBM5 domains in LPMOs have been shown to alter the product profile while acting on crystalline β-chitin substrates [8]. In BcLPMO10A, CBM5 promoted substrate binding as well as protected the enzyme from inactivation [7].

Family Firsts

First indentified

CBM5 modules were first discovered in Endoglucanase, CBDEGZ from Erwinia chrysanthemi [5].

First structural characterization

The first NMR derived structure of CBM5 was from CBDEGZ [5] and first crystal structure was studied for ChBDChiB from Serratia marcescens (SmChiBCBM5) [2].

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 |