Family 6 Carbohydrate Binding Modules Recognize the Non-reducing End of β-1,3-Linked Glucans by Presenting a Unique Ligand Binding Surface

Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the β-1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display β-1,3-glucanase activity. BhCBM6 binds β-1,3-glucooligosaccharides with affinities of ∼1 × 105m-1. The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended β-1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site. Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the β-1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display β-1,3-glucanase activity. BhCBM6 binds β-1,3-glucooligosaccharides with affinities of ∼1 × 105m-1. The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended β-1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site. The plant cell wall represents the largest reservoir of organic carbon in the biosphere, and thus its degradation by microbial enzymes is pivotal to many biological and industrial processes. The plant cell wall is a complex insoluble structure that is highly recalcitrant to biological attack (1Tomme P. Warren R.A. Gilkes N.R. Adv. Microb. Physiol. 1995; 37: 1-81Crossref PubMed Scopus (622) Google Scholar). To increase the efficiency of this degradative process microorganisms synthesize modular plant cell wall hydrolases in which the catalytic modules are attached via linker peptides to non-catalytic carbohydrate-binding modules (CBMs, 1The abbreviations used are: CBM, carbohydrate-binding module; CBM6, family 6 CBM; BhCBM6, family 6 CBM from B. halodurans laminarinase; IMAC, immobilized metal ion affinity chromatography; ITC, isothermal titration calorimetry; MWCO, molecular weight cut-off; r.m.s.d., root mean-square-deviation; MES, 4-morpholineethanesulfonic acid; PDB, Protein Data Bank. 1The abbreviations used are: CBM, carbohydrate-binding module; CBM6, family 6 CBM; BhCBM6, family 6 CBM from B. halodurans laminarinase; IMAC, immobilized metal ion affinity chromatography; ITC, isothermal titration calorimetry; MWCO, molecular weight cut-off; r.m.s.d., root mean-square-deviation; MES, 4-morpholineethanesulfonic acid; PDB, Protein Data Bank. Refs. 2Davies G.J. Henrissat B. Biochem. Soc. Trans. 2002; 30: 291-297Crossref PubMed Google Scholar and 3Boraston A.B. McLean B.W. Kormos J.M. Alam M. Gilkes N.R. Haynes C.A. Tomme P. Kilburn D.G. Warren R.A.J. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. Royal Society of Chemistry, Cambridge1999: 202-211Google Scholar). CBMs potentiate catalytic activity by mediating prolonged and intimate association between the enzyme and its target substrate (4Bolam D.N. Ciruela A. Mcqueen-Mason S. Simpson P. Williamson M.P. Rixon J.E. Boraston A. Hazlewood G.P. Gilbert H.J. Biochem. J. 1998; 331: 775-781Crossref PubMed Scopus (236) Google Scholar, 5Gill J. Rixon J.E. Bolam D.N. Mcqueen-Mason S. Simpson P.J. Williamson M.P. Hazlewood G.P. Gilbert H.J. Biochem. J. 1999; 342: 473-480Crossref PubMed Scopus (69) Google Scholar), while some of these modules also increase substrate access by disrupting the crystalline structure of cellulose (6Din N. Damude H.G. Gilkes N.R. Miller Jr., R.C. Warren R.A. Kilburn D.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11383-11387Crossref PubMed Scopus (155) Google Scholar, 7Din N. Gilkes N.R. Tekant B. Miller R.C. Warren R.A. Kilburn D.G. Bio/Technology. 1991; 9: 1096-1099Crossref Scopus (256) Google Scholar), the major plant structural polysaccharide. CBMs are grouped into 40 sequence-based families (8Coutinho P.M. Henrissat B. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. Royal Society of Chemistry, Cambridge1999: 3-12Google Scholar), which may be found in the continuously updated carbohydrate-active enzyme data base at afmb.cnrs-mrs.fr/CAZY/. These protein modules have also been grouped into three types based on the topology of the binding sites, which reflects the macromolecular structure of the target ligand (3Boraston A.B. McLean B.W. Kormos J.M. Alam M. Gilkes N.R. Haynes C.A. Tomme P. Kilburn D.G. Warren R.A.J. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. Royal Society of Chemistry, Cambridge1999: 202-211Google Scholar, 9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar). Type A CBMs contain a planar hydrophobic ligand binding surface that interacts with crystalline polysaccharides. Type B CBMs, which are derived from 11 different families, contain clefts that accommodate single polysaccharide chains, while the ligand binding sites in Type C CBMs, comprising families 9, 13, 14, 18, and 32, interact with mono- or disaccharides (See Ref. 9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar for review). Type B CBMs generally bind to the substrate hydrolyzed by the appended catalytic domain of the enzyme, leading to considerable variation in ligand specificity within specific families. The three-dimensional structure of almost all Type B CBMs determined to date conform to a classic lectin-like β-jelly roll in which a single ligand binding site comprises a shallow cleft on the concave surface of the protein. In family 4 and family 2 CBM variation in ligand recognition between different members of these families is reflected in the structure of this conserved binding site (10Boraston A.B. Nurizzo D. Notenboom V. Ducros V. Rose D.R. Kilburn D.G. Davies G.J. J. Mol. Biol. 2002; 319: 1143-1156Crossref PubMed Scopus (126) Google Scholar). By contrast family 6 CBMs (CBM6s), which also adopt the classic β-jelly roll fold, contain two potential ligand binding sites; one (cleft B) on the concave surface and a second (cleft A) located on one edge of the protein between the loops that connect the inner and outer β-sheets (11Czjzek M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. Chem. 2001; 276: 48580-48587Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The variation in ligand specificity in CBM6s reflects the functionality of these two binding sites with xylan- and β-(1,4)(1,3) mixed glucan binding modules accommodating these polysaccharides in cleft A and cleft B, respectively (12Pires V.M. Henshaw J. Prates J.A. Bolam D. Ferreira L.M. Fontes C.M. Henrissat B. Planas A. Gilbert H.J. Czjzek M. J. Biol. Chem. 2004; 279: 21560-21568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). One of the features that distinguish Type B CBMs from lectins is the mechanism of ligand recognition. Each binding site in lectins recognizes one or two sugars through an extensive network of hydrogen bonds, while Type B CBMs generally accommodate four to six sugars, with specificity conferred primarily by the conformation of the ligand, which reflects the topology of the binding site. Although it is well established that the orientation of the aromatic residues in the binding site of CBMs confers specificity for the planar and 3-fold helical conformations of cellulose and xylan, respectively (9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar), the mechanism by which these protein modules recognize other polysaccharides, which display more elaborate conformations, is unclear. An example of polysaccharides that exhibit complex conformations is provided by the β-1,3-linked glucose polymer laminarin, which adopts an extensive U-shaped conformation, and thus cannot be accommodated in CBMs that contain linear clefts (9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar). The two potential ligand binding sites in CBM6s may present structural features that make these proteins ideally suited to accommodate polysaccharides with extended U-shaped conformations. To assess this hypothesis we have determined the of a CBM6, BhCBM6, located in an enzyme that displays activity. BhCBM6 bind to affinity for with a the protein displays specificity for the non-reducing of chains, and the crystal structure of BhCBM6 in complex with laminarihexaose that the ligand of cleft A with the surface of the protein that cleft A or cleft B. These data that the in ligand specificity displayed by CBM6s reflects variation in the of the carbohydrate sites on the surface of the and is the of differences in the topology of a single binding site. and and from other and from of and BhCBM6 of the of the the catalytic domain of the enzyme by from halodurans the of Boraston A.B. Alam Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google in The into to The an and a site. The BhCBM6 of the the site is and site is in and into and to The an and a site. and of the and BhCBM6 of the catalytic domain of the in and the protein from by immobilized metal ion affinity the of Bolam D.N. P. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google the protein with BhCBM6 in of as A.B. Alam Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar), and the protein by the of Boraston A.B. Alam Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar). and into in a on a molecular weight by as Chem. Scopus Google The of protein determined by H. Biochem. PubMed Scopus Google of and for the catalytic domain of the and BhCBM6, as A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar, A.B. M. Warren R.A. Kilburn D.G. Biochem. J. 2002; PubMed Scopus Google a with a at between and for and and at the appropriate for The at the and by of the from the and the data against carbohydrate Data for the three with a one site binding for ligand A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar). at in The data are the and of the of three titration as A.B. Alam Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google a in at in the cell and in the which by of into on an of of Ref. A. PubMed Scopus Google Scholar). data the and of two or three of with at and into in a a of BhCBM6 at the from in MES, and of BhCBM6 in complex with by to and these to for of BhCBM6 in complex with laminarihexaose the in with MES, as the Data and the S. D. 1994; PubMed Scopus Google or at a in with at of BhCBM6 in complex with laminarihexaose in the with at Data with a to a x-ray with and an Data the D. Biol. 1999; PubMed Scopus Google provided with the In all data of the as PubMed Scopus Google and used to In the of the BhCBM6 and the as are in for and data used in the structure and and structure of data in are for the high protein and and and of of of sugar 4 in are for the high in a the data for the and the of the CBM6 from the Ref. M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. Chem. 2001; 276: 48580-48587Abstract Full Text Full Text PDF PubMed Scopus (98) Google as a the A. A. D. Biol. PubMed Scopus Google to two molecular to the two BhCBM6 in the One by of J. Biol. 1999; PubMed Scopus Google and with D. PubMed Scopus Google Scholar). This used to the second in the cell by of and This used as a in the and of the the used to the structure of the laminarihexaose This and the laminarihexaose in by with and to are in and with and are in The BhCBM6, complex and complex have been with the of and the interactions of BhCBM6 with and laminarihexaose to in the are the with and are in with The conserved in the cleft A binding site of CBM6s is with an of cleft A of BhCBM6 in complex with laminarihexaose from in complex with Ref. V.M. Henshaw J. Prates J.A. Bolam D. Ferreira L.M. Fontes C.M. Henrissat B. Planas A. Gilbert H.J. Czjzek M. J. Biol. Chem. 2004; 279: 21560-21568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and from in complex with Ref. A. Boraston A.B. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). residues are as and and and in BhCBM6, and The which in BhCBM6, also the comprising residues that is in the The and of the B. halodurans halodurans an that a of on J. Miller PubMed Scopus Google the protein to three modules The of this protein similarity with family a family of proteins in which some members display β-1,3-glucanase by from which is in cell V. S. A.B. 2002; PubMed Scopus Google Scholar, A.B. M. J. Sci. PubMed Scopus Google Scholar), an example of which is the are of catalytic activity but bind J. A. J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The catalytic from the B. halodurans protein is the example in this family. The catalytic of this enzyme hydrolyzed with an activity of of sugar of enzyme but displays activity against xylan, cellulose, or the B. halodurans protein is a β-1,3-glucanase to its The to proteins of the and the catalytic is a of to the binding CBM6 from (11Czjzek M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. Chem. 2001; 276: 48580-48587Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). on its with CBM6s and its in a we that this as BhCBM6, is a CBM with β-1,3-glucan binding of BhCBM6 a binding B.W. Boraston A.B. Gilkes N.R. Haynes C.A. Kilburn D.G. Protein PubMed Google binding to the insoluble polysaccharides cellulose or insoluble β-1,3-glucan from affinity binding to of or the two of which are by β-1,3-glucan binding CBMs A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, J. Bolam D.N. V.M. Czjzek M. Henrissat B. Ferreira L.M. Fontes C.M. Gilbert H.J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF Scopus Google Scholar). a of the of Gilbert H.J. Bolam D.N. Boraston A.B. Biochem. 2004; PubMed Scopus Google BhCBM6 binding to and but interact with polysaccharides of BhCBM6 and in the binding to these The of and β-1,3-glucooligosaccharides also for a at with the to binding to by affinity and of the or to in the used to the of the on ligand and binding to BhCBM6 to glucose and with association of × more a of binding on ligand in affinity the ligand to and with affinities to that for The β-1,3-glucooligosaccharides and laminarihexaose with association from ∼1 × to ∼1 × the of BhCBM6 for β-1,3-glucooligosaccharides and the of affinity on sugar the to binding to polysaccharides β-1,3-linked glucose residues by affinity and BhCBM6 to be primarily a CBM, with its in a of BhCBM6 for sugars determined by at in in a of titration the of BhCBM6 with β-1,3-glucooligosaccharides to be and at interactions to The the of a protein to carbohydrate macromolecular CBMs that bind to extended B CBMs, Ref. 9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google a increase in binding affinity with sugar BhCBM6 a in affinity with the affinity from to laminarihexaose The of affinity binding to be by an that more a in The for this is in of the structure of BhCBM6 in complex with laminarihexaose which in how this third sugar interacts with the of BhCBM6 for sugars determined by isothermal titration at in in a binding to by in a where the protein into the polysaccharide. The binding which highly with a single of binding two binding one at to and at A of these which the of the These multiple of binding sites present in the or in the cannot on this and of the complex of recognition the and of binding for the interactions be from the of the association in the range of with the affinities for The of binding sites by of the of the in the in of and This an of ∼1 CBM which is with BhCBM6 binding to the ends of The binding to and to by this of BhCBM6 to into the mechanism of carbohydrate recognition by BhCBM6 we its three-dimensional structure by x-ray and The of BhCBM6 in the of ligand of two BhCBM6 six two and are in other CBM6s structures have been BhCBM6 adopts a with a a This highly to the CBM6 from a of (11Czjzek M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. Chem. 2001; 276: 48580-48587Abstract Full Text Full Text PDF PubMed Scopus (98) Google the two CBM6s from a of and A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies J. Mol. Biol. PubMed Scopus Google Scholar, A. Boraston A.B. J. Mol. Biol. 2004; PubMed Scopus Google and the glucan binding CBM6 from a of (12Pires V.M. Henshaw J. Prates J.A. Bolam D. Ferreira L.M. Fontes C.M. Henrissat B. Planas A. Gilbert H.J. Czjzek M. J. Biol. Chem. 2004; 279: 21560-21568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Each BhCBM6 metal The is by the of and The is by the of and a single The of this metal ion is conserved with the four other CBM6 where these metal as in the of BhCBM6, the to an high this as a metal with on the of a high of in the used to BhCBM6 the to the metal ion as The second metal also as by the of and The of and a also in binding this The third ion is to the protein by one of and four is while the is as its to be conserved in this protein the of the other two is unclear. BhCBM6 in with complex of BhCBM6 by in for two to of the two of BhCBM6 in the cell The binding site a sugar The in this site interactions with the with one a potential hydrogen with a second BhCBM6 the biological of this binding site is and be The other binding which accommodated a in the is the conserved binding site binding The for the to this site of the BhCBM6 of all of the in this of the to between the of and the of the sugar non-reducing This to some by the of and one orientation in in the other two well to be to hydrogen to to be the in of the residues based on the the carbohydrate as the non-reducing sugar between and while the hydrogen reveals potential hydrogen potential hydrogen are present with one potential interactions and is conserved with in other CBM6s and The to bind in this orientation is by the of to interact with This with a end, its that BhCBM6 be to accommodate the non-reducing of the we have that BhCBM6 cannot also bind the of is that the non-reducing of the sugar is in the binding such that the cannot a wall of the binding to the In the to into BhCBM6 in with with laminarihexaose in the with a single protein and sugar in the six glucose residues of the laminarihexaose be The glucose at the non-reducing of this between and as for The complex of BhCBM6 with laminarihexaose six binding The extended of this binding site and for with a of this CBM as a Type B CBM (9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar). potential hydrogen these with the of hydrogen with other Type B CBMs (9Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-782Crossref PubMed Scopus (1473) Google Scholar). potential hydrogen by The laminarihexaose adopts a to that in the x-ray crystal structure of the CBM from a 6 and Ref. A.B. Nurizzo D. Notenboom V. Ducros V. Rose D.R. Kilburn D.G. Davies G.J. J. Mol. Biol. 2002; 319: 1143-1156Crossref PubMed Scopus (126) Google and to that for in V. S. PubMed Scopus Google Scholar). to the the of interactions between BhCBM6 and laminarihexaose are unique to the conformation of this sugar and against high affinity for other the family 4 CBM, which binds in a binding the BhCBM6 binding site with a to accommodate the glucose at the of the The of the the CBM to a The or conformation of the ligand to the that the binding site and interactions with the protein. In polysaccharides that have a or 3-fold linear such as cellulose and xylan, into and make interactions with the protein displays the affinity for the protein as and increase in affinity is for The sugar at the non-reducing of laminarihexaose interacts with the of the CBM6 binding site such that is at the protein surface extension of the sugar polymer and thus conferring specificity for the non-reducing of the polysaccharide. of CBM6 to the in of the structures of CBM6 modules that recognize and mixed β-(1,4)(1,3) with BhCBM6, which binds to laminarin, into the structural for the extensive range of ligand specificities displayed by this family of In the binding CBM6s cleft A is at ends these proteins are to bind to the of the with the sugar in this site between two aromatic residues The surface of the cleft is to with the of the at 4 and against binding to By the ligand binding site in the CBM6 that recognizes β-(1,4)(1,3) mixed is in cleft B, which the extended that this binding site in the other CBM6 A in the CBM display affinity for the sugars of and but the of the polysaccharides. The is between the aromatic residues but is to the of the sugar in the binding modules and thus is at the surface of the protein and sugars attached to into and thus interact with the protein The topology of cleft A in BhCBM6 is to the CBM A which at this is an or in binding one of the binding site in the CBM6 and while in the CBM this hydrogen with of the in BhCBM6 it interacts with the non-reducing The unique feature of the BhCBM6 binding site is an extended comprising residues This and the of a that the U-shaped of the and of cleft A and a surface that is distinct from the cleft A of CBM6s the specificity of BhCBM6 for is conferred by a binding surface that is different from cleft A and cleft B, but a while the ligand binding site of all other Type B CBMs conform to concave These data therefore a third ligand binding site in the CBM6 family of proteins that a unique These data how the multiple binding clefts and highly unusual protein surface of CBM6s confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members is conferred by differences in the topology of a single binding while the range of ligand recognition in CBM6 is the of variation in the of the ligand binding site in different members of this family. biological for the of BhCBM6 to the non-reducing ends of β-1,3-glucan is and as the of binding sites be for the of Type B CBMs, which bind to the of polysaccharides. but to the of plant structural polysaccharides is by from the A.B. Alam Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar, V. Boraston A.B. Kilburn D.G. Rose D.R. 2001; PubMed Scopus Google Scholar). it is that of the B. halodurans to the ends of polysaccharide may an of action by the enzyme, the by its catalytic domain are with a of the enzyme that display a range of different hydrolases a single The of B. halodurans to the ends of may the of the macromolecular structure that this as a is recalcitrant to is that of the plant cell through or the action of other contain a of polysaccharide and be to BhCBM6, by the enzyme to these may potentiate its catalytic activity.

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