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Carbohydrate Binding Module Family 6
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The ligand specificity of the first characterized CBM6, originating from a multimodular xylanase from Clostridium thermocellum, was determined to be xylan , although the results showed that this CBM6 was also able to bind to avicel and acid-swollen cellulose. This was also the first CBM6 for which a 3D structure was determined , and multiple sequence alignments, analyzed in the light of the first 3D structure, already gave clear indications that large diversity in specificity was to be expected among CBM6 modules . The first ligand-bound complex of a xylanase CBM6 (CsCBM6-3) from Clostridium stercorarium was crystallized by Boraston et al. . Remarkably, the characterization and 3D structure of a CBM6 from Cellvibrio mixtus revealed two distinct binding sites that displayed differential binding specificities [4, 5]. CBM6 modules are in general attached to bacterial or archeal polysaccharide degrading enzymes and can be found attached to xylanases, cellulases, agarases, laminarinases, etc . Interestingly, modules assigned to the CBM6 family have also been found associated to fungal enzymes and to the α-subunit of the coagulation factor G in horseshoe crabs (see the occurance of CBM6 in eukaryotes). In the latter case, the β-1,3-glucan binding of the C-terminal tandem CBM6s has been demonstrated . These CBM6s have characterized binding specificities for linear and branched/decorated xylan, β-1,4-glucan (or cellulose), mixed-linked β-1,3-1,4-glucan (or lichenan), agarose, β-1,3-glucan (or laminarin) or chitin. Based on phylogenetic analyses of all reported CBM6 sequences in 2009 (a total of 167), four subfamilies have been defined that coincide with classes of substrate binding specificity as follows : subfamily 6a, hemicellulose; subfamily 6b, xylan; subfamily 6c, β-glucans with a variety of linkages; and subfamily 6d, agarose .
Like the majority of CBMs, CBM6s display the typical β-sandwich fold predominantly consisting of five antiparallel β-strands on one face and four anti-parallel β-strands on the other face, connected by loops with variable lengths. Within the hierarchical CATH classification the modules belong to the jelly-roll superfamily 188.8.131.520 called "galactose-binding domain-like" that contains 515 unique domains. The first identified ligand binding site was not located at a shallow cleft on the concave surface of the β-sheets (binding site II, formerly called cleft B in CBM6) as was typically observed in CBMs. Alternatively, a binding site was found located at the apex, within the connecting loops of the two β-sheets (binding site I, formerly cleft A in CBM6) (PDB 1gmm). Due to the existence of the dual binding sites in CBM6s, both Type B and C binding properties have been observed for individual CBM6s. Interestingly, some CBM6s display binding affinities for both binding sites (PDB 1uyy), either with distinct specificities for each site (PDB 1uy0 and PDB 1uyz) or synergistic binding involving both sites at the same time , while binding properties of other CBM6s make use of only one binding site, which is in general site I at the apex (i.e. PDB 1uxx;PDB 1nae;PDB 1w9w). The apex site I is made up of two important, highly conserved aromatic residues (mostly W and Y) that "sandwich" a sugar monomer [2, 8]. These conserved residues are neighboured by a much more variable loop (defined as zone E in Abbott et al. ) that make up the diversity in binding specificity. Consistently, a variable number of sugar-binding subsites have been observed for site I, ranging from one (end binder) up to five binding subsites. The precise structural and energetic contributions of four of the binding subsites have been dissected, for the first time, in detail by combining crystallography and isothermal titration calorimetry (ITC) in the case of the Clostridium stercorarium CsCBM6-1 . To date, only one CBM6 has been structurally and biochemically characterized that makes use of binding site II, which is the CBM6 from Cellvibrio mixtus [4, 5] (PDB 1uxz). An updated list of all available three-dimensional structures is accessible at the Cazy CBM6 structures page.
The predominant functional role to date described for CBM6 modules is carbohydrate binding and targeting. It has been shown that this type of module synergistically enhances the activity of the adjacent catalytic domain on insoluble substrates. The most common associated modules are enzymes such as xylanases, lichenases, β-agarases, laminarinases, deacetylases; other modules that have been found associated to CBM6 are dockerins and in eukaryotic organisms coagulation factors. The CBM6 from Clostridium thermocellum has been used to label plant tissues .
- First Identified
- The xylose binding CBM6 from a multi-modular xylanase/actetyl-esterase from Clostridium thermocellum (CtCBM6) was the first to be identified and biochemically characterized. To a lesser extent the module was also able to bind to avicel and acid-swollen cellulose .
- First Structural Characterization
- The same xylose binding CBM6 from Clostridium thermocellum (PDB 1gmm) was also the first structurally characterized CBM6. The 3D structure revealed that the location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence was not conserved . The first CBM6 in complex with its ligand was determined for the CsCBM6-3 from Clostridium stercorarium (PDB 1o8p).
- Fernandes AC, Fontes CM, Gilbert HJ, Hazlewood GP, Fernandes TH, and Ferreira LM. (1999) Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem J. 342 ( Pt 1), 105-10.
- Czjzek M, Bolam DN, Mosbah A, Allouch J, Fontes CM, Ferreira LM, Bornet O, Zamboni V, Darbon H, Smith NL, Black GW, Henrissat B, and Gilbert HJ. (2001) The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved. J Biol Chem. 276, 48580-7. DOI:10.1074/jbc.M109142200 |
- Boraston AB, Notenboom V, Warren RA, Kilburn DG, Rose DR, and Davies G. (2003) Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains. J Mol Biol. 327, 659-69. DOI:10.1016/s0022-2836(03)00152-9 |
- Henshaw JL, Bolam DN, Pires VM, Czjzek M, Henrissat B, Ferreira LM, Fontes CM, and Gilbert HJ. (2004) The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities. J Biol Chem. 279, 21552-9. DOI:10.1074/jbc.M401620200 |
- Pires VM, Henshaw JL, Prates JA, Bolam DN, Ferreira LM, Fontes CM, Henrissat B, Planas A, Gilbert HJ, and Czjzek M. (2004) The crystal structure of the family 6 carbohydrate binding module from Cellvibrio mixtus endoglucanase 5a in complex with oligosaccharides reveals two distinct binding sites with different ligand specificities. J Biol Chem. 279, 21560-8. DOI:10.1074/jbc.M401599200 |
- Michel G, Barbeyron T, Kloareg B, and Czjzek M. (2009) The family 6 carbohydrate-binding modules have coevolved with their appended catalytic modules toward similar substrate specificity. Glycobiology. 19, 615-23. DOI:10.1093/glycob/cwp028 |
- Takaki Y, Seki N, Kawabata Si S, Iwanaga S, and Muta T. (2002) Duplicated binding sites for (1-->3)-beta-D-glucan in the horseshoe crab coagulation factor G: implications for a molecular basis of the pattern recognition in innate immunity. J Biol Chem. 277, 14281-7. DOI:10.1074/jbc.M200177200 |
- Abbott DW, Ficko-Blean E, van Bueren AL, Rogowski A, Cartmell A, Coutinho PM, Henrissat B, Gilbert HJ, and Boraston AB. (2009) Analysis of the structural and functional diversity of plant cell wall specific family 6 carbohydrate binding modules. Biochemistry. 48, 10395-404. DOI:10.1021/bi9013424 |
- Lammerts van Bueren A and Boraston AB. (2004) Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites. J Mol Biol. 340, 869-79. DOI:10.1016/j.jmb.2004.05.038 |
- McCartney L, Gilbert HJ, Bolam DN, Boraston AB, and Knox JP. (2004) Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers. Anal Biochem. 326, 49-54. DOI:10.1016/j.ab.2003.11.011 |