The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

Abstract

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display β-1,4- and β-1,3–1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both β-1,4- and β-1,3–1,4-mixed linked glucans, displaying Ka values of 1.9 × 105, 4.4 × 104, and 2 × 103m-1 for Glc-β1,4-Glc-β1,4-Glc-β1,3-Glc, Glc-β1,4-Glc-β1,4-Glc-β1,4-Glc, and Glc-β1,3-Glc-β1,4-Glc-β1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 Å. The protein displays a β-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr22, Tyr53, and Tyr129, located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both β-1,4- and β-1,3–1,4-mixed linked glucans. Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display β-1,4- and β-1,3–1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both β-1,4- and β-1,3–1,4-mixed linked glucans, displaying Ka values of 1.9 × 105, 4.4 × 104, and 2 × 103m-1 for Glc-β1,4-Glc-β1,4-Glc-β1,3-Glc, Glc-β1,4-Glc-β1,4-Glc-β1,4-Glc, and Glc-β1,3-Glc-β1,4-Glc-β1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 Å. The protein displays a β-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr22, Tyr53, and Tyr129, located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both β-1,4- and β-1,3–1,4-mixed linked glucans. The major plant cell wall polysaccharides, cellulose and hemicellulose, are hydrolyzed into soluble sugars by a consortium of microbial enzymes. In nature, these enzyme systems play a major role in recycling plant cell wall fixed carbon and are therefore of considerable biological and biotechnological importance. In general, glycoside hydrolases (GH) 1The abbreviations used are: GH, glycoside hydrolase; CBM, carbohydrate-binding module; ITC, isothermal titration calorimetry. that catalyze plant cell wall degradation contain noncatalytic carbohydrate-binding modules (CBMs) that interact with polysaccharides (1Gilbert H.J. Bolam D.N. Szabo L. Xie H. Williamson M.P. Simpson P.J. Jamal S. Boraston A.B. Kilburn D.G. Warren R.A. Teeri T.T. Svensson B. Gilbert H.J. Feizi T. Carbohydrate Bioengineering: Interdisciplinary Approaches. The Royal Society of Chemistry, Cambridge, UK2001: 89-98Google Scholar). Based on primary structure similarity, CBMs have been grouped into 39 different families (afmb.cnrs-mrs.fr/CAZY) (2Coutinho P.M. Henrissat B. Gilbert H.H. Davies G. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge, UK1999: 3-12Google Scholar). By mediating an intimate and prolonged association of the enzyme with its target substrate, CBMs enhance the activity of the catalytic module against insoluble polysaccharides (3Gill 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, 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 (237) Google Scholar). Therefore, CBMs play a major role in potentiating the capacity of cellulases and hemicellulases to degrade plant cell wall polysaccharides. Structural studies have revealed that the topology of the ligand-binding site of CBMs varies. CBMs that interact with the flat surfaces of crystalline polysaccharides contain a planar hydrophobic carbohydrate-binding site and are referred to as Type A CBMs (5Kraulis J. Clore G.M. Nilges M. Jones T.A. Petterson G. Knowles J. Gronenborn A.M. Biochemistry. 1989; 28: 7241-7257Crossref PubMed Scopus (489) Google Scholar, 6Raghothama S. Simpson P.J. Szabo L. Nagy T. Gilbert H.J. Williamson M.P. Biochemistry. 2000; 39: 978-984Crossref PubMed Scopus (68) Google Scholar, 7Tormo J. Lamed R. Chirino A.J. Morag E. Bayer E.A. Shoham Y. Steitz T.A. EMBO J. 1996; 15: 5739-5751Crossref PubMed Scopus (414) Google Scholar, 8Xu G.Y. Ong E. Golkes N.R. Kilburn D.G. Muhandiram D.R. Harris-Brandts M. Carver J.P. Kay L.E. Harvey T.S. Biochemistry. 1995; 34: 6993-7009Crossref PubMed Scopus (214) Google Scholar). In contrast, CBMs that bind to single polysaccharide chains accommodate these ligands in extended clefts and are defined as Type B CBMs (9Charnock S.J. Bolam D.N. Turkenburg J.P. Gilbert H.J. Ferreira L.M. Davies G.J. Fontes C.M. Biochemistry. 2000; 39: 5013-5021Crossref PubMed Scopus (151) Google Scholar, 10Boraston A.B. Revett T.J. Boraston C.M. Nurizzo D. Davies G.J. Structure. 2003; 11: 665-675Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 11Szabo L. Jamal S. Xie H. Charnock S.J. Bolam D.N. Gilbert H.J. Davies G.J. J. Biol. Chem. 2001; 276: 49061-49065Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Boraston 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 (128) Google Scholar). Type B CBMs have been described that bind to a diversity of ligands, with some modules displaying plasticity in their capacity to accommodate heterogeneity in the sugar backbone in terms of both the identity of the saccharides and the nature of the linkage. Thus, polar residues in CBM29 are able to hydrogen bond to the axial O2 in mannose and the equatorial O2 in glucose, enabling the protein to bind cellulose, glucomannan, and mannan (13Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (84) Google Scholar). Some CBMs from families 4 and 6 display affinity for the β-1,3–1,4-mixed linked glucans (barley β-glucan and lichenan) and β-1,4 glucans such as amorphous cellulose and cellohexaose (14Czjzek 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 (100) Google Scholar, 15Boraston A.B. Warren A.J. Kilburn D.G. Biochemistry. 2001; 40: 14679-14685Crossref PubMed Scopus (32) Google Scholar). It is currently unclear whether these proteins interact exclusively with the β-1,4 regions of the mixed linked polysaccharide or display specificity for sequences of glucose units that are linked by a mixture of β-1,3- and β-1,4-glycosidic bonds. In general the ligand specificity of Type B CBMs reflects the substrate hydrolyzed by the associated catalytic modules (1Gilbert H.J. Bolam D.N. Szabo L. Xie H. Williamson M.P. Simpson P.J. Jamal S. Boraston A.B. Kilburn D.G. Warren R.A. Teeri T.T. Svensson B. Gilbert H.J. Feizi T. Carbohydrate Bioengineering: Interdisciplinary Approaches. The Royal Society of Chemistry, Cambridge, UK2001: 89-98Google Scholar). In enzymes that contain multiple catalytic domains with distinct substrate specificities, however, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To investigate whether CBMs can display enhanced affinity for mixed linked glucans and to interrogate the evolutionary pressure exerted on the ligand specificity of CBMs in enzymes that contain distinct catalytic modules, we have studied the structure and function of the family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E (16Yague E. Beguin P. Aubert J.P. Gene (Amst.). 1990; 89: 61-67Crossref PubMed Scopus (66) Google Scholar). This enzyme contains GH5 and GH26 catalytic domains that display β-1,4 and β-1,3–1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 does indeed display a preference for specific β-1,3–1,4-mixed linked glucans, although the protein is able to bind to β-1,4-glucose polymers. The three-dimensional structure of CtCBM11 in harness with mutagenesis studies reveals that the protein contains a single ligand-binding cleft that can accommodate both β-1,3–1,4- and β-1,4-linked glucans. Protein Expression and Purification—To express CtCBM11 in Escherichia coli, the region of the Lic26A-Cel5A gene (lic26A-cel5A) encoding the internal family 11 CBM was amplified from C. thermocellum YS genomic DNA using the thermostable DNA polymerase pFU Turbo (Stratagene). The primers used, 5′-CTCGCTAGCGCTGTCGGTGAAAAAATG-3′ and 5′-CACCTCGAGAGCACCAATCAGCTTGAT-3′, incorporated NheI and XhoI restriction sites, which are depicted in bold. The PCR product was cloned into pGEM T-easy (Promega) and sequenced to ensure that no mutations had occurred during the polymerase chain reaction. The recombinant pGEM T-easy derivative was digested with NheI and XhoI, and the excised CtCBM11 encoding gene was cloned into the similarly restricted expression vector to CtCBM11 by contains a E. was in to which was to a of and the for a The by and the cell was in a and recombinant CtCBM11 was by affinity as described Nagy T. Gilbert H.J. Davies G.J. Ferreira L.M. Fontes C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). of soluble polysaccharides from and which from from was from cellulose was as described Scopus Google Scholar). The mixed linked and by the of M. O. A. Biochemistry. 2003; PubMed Scopus Google Scholar). affinity of CtCBM11 for a of soluble polysaccharides was by affinity The was as described by P. Boraston A. Warren A.J. Kilburn D.G. 2000; PubMed Scopus Google using the polysaccharide ligands a of was for 4 in The was of binding was as described Scopus Google Scholar). to of CtCBM11 binding to and cellulose was as of protein in and mixed with of ligand in a of The mixture was for 2 4 with which the insoluble ligand was by × for The the was and the was with of A. The protein was by the polysaccharides in of for and by using a protein no polysaccharide in to ensure that no for all ligands which was to determine the D.N. Xie H. G. D. G. Henrissat B. Gilbert H.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (69) Google using a titration The proteins against and the ligand was in the same to of a titration the protein in a cell was with of polysaccharide or ligand The of sites in the polysaccharide ligands was as described L. Jamal S. Xie H. Charnock S.J. Bolam D.N. Gilbert H.J. Davies G.J. J. Biol. Chem. 2001; 276: 49061-49065Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). for of by using a binding The the association of binding sites on the protein and the of binding using the in for ligands, and the are the of the of these Site-directed of CtCBM11 using the mutagenesis to the The primers used to these as and and and and and The DNA sequences sequenced by to ensure that the mutations had been incorporated into the and of E. with was in of as described by Charnock (9Charnock S.J. Bolam D.N. Turkenburg J.P. Gilbert H.J. Ferreira L.M. Davies G.J. Fontes C.M. Biochemistry. 2000; 39: 5013-5021Crossref PubMed Scopus (151) Google Scholar). Expression of CtCBM11 was by the of The for a which and the recombinant protein was by affinity as described was as a The enzyme was using a into to with and to using a with protein in A. enzyme was as described and with using the same and the protein was to and of protein by using the with an of protein in and and a of and in a and The in using as to the A of derivative crystal was on the The crystal was by using a The CtCBM11 crystal to with cell a and which to a of and a of The with the 276: PubMed Scopus Google and the are in and structure of is the of and is the from multiple of of protein of used in × and are the and structure is for a of the B is the of and is the from multiple of × and are the and structure is for a of the in a and sites located and with Biol. 1999; PubMed Scopus Google using the and the which a of This in to 1.98 with a of of which by with and on Protein Scholar). using A. R. Biol. 1999; PubMed Scopus Google a with This of of a of residues and the CtCBM11 primary with A. C. with in Biol. PubMed Scopus Google and of the CtCBM11 primary in a with This residues of which are and and to a in the as as and of the and residues of the of the of family 11 CBM of endoglucanase of was to bind L.M. T. PubMed Google Scholar). It was on this that of this family as To the ligand binding specificity of CtCBM11, the protein module was as a in E. and to The affinity of CtCBM11 for glucomannan, and cellulose, and was by affinity The that CtCBM11 displays the affinity for binding to cellulose, and CtCBM11 with or of CtCBM11 and its to soluble polysaccharides by affinity values are as the ligand Ka The values are as the ligand Ka in a To the ligand specificity of CtCBM11 in the binding of the protein to polysaccharides and was using in titration for β-1,3–1,4- and as as the mixed linked polysaccharides β-glucan and enabling the and of binding to for these ligands The show that CtCBM11 displays affinity for and the protein affinity for × binding to is to by × that CtCBM11 contains sites, which is consistent with the ligands accommodated by Type B the protein displays affinity for the β-1,3–1,4-mixed linked that the CBM displays a preference for a glucose in To investigate this bond preference in the capacity of CtCBM11 to bind a of was The protein no affinity for a of glucose, that all the sites in the CBM can accommodate glucose residues CtCBM11, however, to the mixed the that the protein displays a preference for a glucose in A mixed Glc-β1,3-Glc-β1,4-Glc-β1,3-Glc, to CtCBM11 × affinity to by that the protein able to accommodate a single The ligand specificity of CtCBM11 is consistent with the that glucose polymers mixed of and have the of and contain for with Glc-β1,4-Glc-β1,4-Glc-β1,3-Glc, and that are linked by bonds. Thus, the of specificity for is consistent with their from the target polysaccharides, of mixed linked The binding of CtCBM11 to its ligands is associated with and The of the of CtCBM11 with glucose polymers is of the binding of proteins to soluble which is with an The capacity of CtCBM11 for revealed a of values for are associated with the of surfaces ligand This is consistent with the role play in the of CtCBM11 with all its ligands and of CtCBM11 binding to and polysaccharides as by × is the of binding sites on the values are with the of is the of binding sites on the The values are with the of in a The ligand specificity of CtCBM11 the substrate specificity of the enzyme Lic26A-Cel5E, which displays both β-1,3–1,4-mixed linked and β-1,4-linked endoglucanase activity, by the and catalytic modules, respectively. J. and G. J. in It however, that Lic26A-Cel5E is a of a C. thermocellum the that contains enzymes and Y. Lamed R. Bayer E.A. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the properties of CtCBM11 exclusively the of the activities by Lic26A-Cel5E the ligands accommodated by the module bond by enzymes the that display substrate specificities to and It however, that in enzymes the the specificities of CBMs are to the substrate specificity of the appended catalytic domains. Thus, the ligands recognized by CBMs in C. thermocellum enzymes are by their the of the that the specificity of these modules is by the substrates hydrolyzed by the of CtCBM11 and with structure of the CtCBM11 was solved using with a derivative to a resolution of 1.98 which of for of the and the are in The CtCBM11 of residues from a of and from the of The and and The CtCBM11 structure reveals a of which a side and and a concave side and and a The of the is hydrophobic and and The of the is in is by side chain of from and and and the chain of was in and a binding site was in and is to residues and chain and and the The the ligands and the from to sites of of the in CtCBM11 in the The residues in binding are as and The chain are in and the are as The with the A. C. to CtCBM11, of CBM families and bind these are and are to the protein V. Boraston A.B. P. Kilburn D.G. Rose D.R. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The of in the of a family 4 CBM, which contains binding sites for the was by that of the from the module the by M. Simpson P.J. S. P. Williamson M.P. S.J. Gilbert H.J. Bolam D.N. O. Biochemistry. 2002; PubMed Scopus Google Scholar). it was that in CBMs from families and a role in ligand binding D.N. Xie H. G. D. G. Henrissat B. Gilbert H.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (69) Google Scholar, S. Boraston A.B. Turkenburg J.P. Ducros Davies G.J. Structure. Scholar). The of the sites in CtCBM11, however, is from the carbohydrate-binding cleft, a role for the in CBMs that bind chains, the concave side of CtCBM11 forms a cleft. In CtCBM11, this surface is defined by and and the in 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 (128) Google Bolam D.N. Ferreira L.M. Fontes C.M. Henrissat B. A. Gilbert H.J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google L. Jamal S. Xie H. Charnock S.J. Bolam D.N. Gilbert H.J. Davies G.J. J. Biol. Chem. 2001; 276: 49061-49065Abstract Full Text Full Text PDF PubMed Scopus (85) Google V. Boraston A.B. P. Kilburn D.G. Rose D.R. J. Mol. Biol. 2001; PubMed Scopus Google (9Charnock S.J. Bolam D.N. Turkenburg J.P. Gilbert H.J. Ferreira L.M. Davies G.J. Fontes C.M. Biochemistry. 2000; 39: 5013-5021Crossref PubMed Scopus (151) Google Scholar, H. Gilbert H.J. Charnock S.J. Davies G.J. Williamson M.P. Simpson P.J. S. Fontes C.M. Ferreira L.M. Bolam D.N. Biochemistry. 2001; 40: PubMed Scopus Google A.B. Revett T.J. Boraston C.M. Nurizzo D. Davies G.J. Structure. 2003; 11: 665-675Abstract Full Text Full Text PDF PubMed Scopus (60) Google and CBM29 (13Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (84) Google Scholar). The side chains of Tyr22, Tyr53, Tyr129, and and the cleft of a of the to this cleft is by the residues and of a to the forms hydrogen with residues and the side chain of the of and Tyr129, which are and are by from the of the same a hydrogen bond with an of of the residues that the to the cleft. The cleft to the carbohydrate-binding site in the with residues Tyr22, Tyr53, Tyr129, and a role in ligand A for proteins to CtCBM11 was using the L. C. J. Mol. Biol. PubMed Scopus Google Scholar). The show that CtCBM11 is to carbohydrate-binding domains from different the of family CBM from the of from the family 4 CBM from the family CBM from C. thermocellum the family CBM from the family CBM from T. the ligand-binding of the from the family CBM from Clostridium and the family 6 CBM from all a and the are restricted to The of these on CtCBM11 in the of Å. A single site is in the of and from T. and from C. and from C. and this to in CtCBM11, although the ligands In all of these the carbohydrate-binding cleft is and from the of the CtCBM11 a cleft, is by and In the of and this of the cleft is in CtCBM11, of It is that the β-sandwich by CtCBM11 is the CBMs and that families and with the CBM family 11 module described a as by A. Biochem. J. 2001; PubMed Scopus Google Scholar). is a that all of these CBM families a evolutionary that in evolutionary catalytic modules have the noncatalytic CBMs, the glycoside hydrolases are for the of the β-sandwich of A in the catalytic such as the of the in a GH, a CBM. it is that can the ligand specificity of CBMs, and therefore the of mutations have for the evolution of the ligand specificities by the enzymes P.J. Xie H. Bolam D.N. Gilbert H.J. Williamson M.P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (128) Google Scholar). CtCBM11 to glycoside hydrolases from of from the of from and from In the structure from S. G. C. Davies G.J. Biochemistry. 1999; PubMed Scopus Google a glucose was in the carbohydrate-binding cleft linked to a a and are to the of described of the surface of the cleft of CtCBM11 reveals the of which are in the these is in all Tyr22, Tyr53, and are in of the sequences in this of of residues in ligand binding is the plasticity of specificities in Type B CBM it is that all of the residues are in CtCBM11 and interact with the target ligands hydrophobic To the of residues Tyr22, Tyr53, Tyr129, and in ligand the proteins and and their properties with The and show that no affinity for the ligands and binding for these polysaccharides. The in the of the against all the ligands, with CtCBM11, that these residues play an role in the recognition of glucomannan, and β-1,4- and glucans. By contrast, displays affinity to CtCBM11 for these soluble ligands, that does to ligand binding to the capacity of CtCBM11 to interact with insoluble polysaccharides. The in that the family 11 CBM of Lic26A-Cel5A is able to interact with and The and display binding to the insoluble ligands, that cellulose binds to the the extended cleft. The of the cleft binding to the planar of crystalline regions of cellulose, and the CBM is to interact with single cellulose chains in the regions of the forms of The of to had no on binding to the insoluble these that the located in CtCBM11 is the carbohydrate-binding cleft and that residues Tyr22, Tyr53, and play a major role in the of CtCBM11 with its Structural studies of family 6 CBMs which are to the that these proteins contain and different clefts that display different ligand specificities Bolam D.N. Ferreira L.M. Fontes C.M. Henrissat B. A. Gilbert H.J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the binding clefts in from C. endoglucanase was to interact with β-1,3–1,4-mixed linked and β-1,4-linked glucans with binding to Bolam D.N. M. Henrissat B. Ferreira L.M. Fontes C.M. Gilbert H.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The crystal structure of that in cleft a β-1,4 glucose can accommodated the can interact with β-1,4- or glucose the displays specificity for a glucose Bolam D.N. Ferreira L.M. Fontes C.M. Henrissat B. A. Gilbert H.J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the CtCBM11 with the mutagenesis that the family 11 module contains a single ligand-binding in with the of CBMs characterized to In the preference by CtCBM11 a general of CBMs that recognize β-1,3–1,4-mixed linked glucans. Thus, the capacity of the Clostridium CBM to bind β-1,4- and ligands reflects plasticity a single binding site the of multiple carbohydrate-binding affinity with β-glucan as the CtCBM11 and to in a of different of The of the soluble polysaccharide in the and of CtCBM11 and its to insoluble polysaccharides as by Protein was mixed with and cellulose and for 2 4 protein was and the was for in and protein was by in The of the and are in reveals that CtCBM11 binds to glucans displaying considerable affinity for β-1,4-linked glucose polymers and no affinity for glucans. This ligand specificity reflects the substrate specificity of the associated GH26 and GH5 catalytic modules that on β-1,3–1,4- or The crystal structure of CtCBM11 with mutagenesis show that the in the protein structure the ligand-binding site and that residues Tyr22, Tyr53, and play a central role in and are located to in CtCBM11 cleft and are in all and are to against the of a single sugar Tyr22, however, is in of the it is that this of reflects in ligand specificity in modules, it is that of a in the affinity for polysaccharides and The in this that the different substrate specificities by the catalytic domains in Lic26A-Cel5E both influence the ligand specificity of the appended We that CBMs can display specificity for of glucose residues that are linked by a specific of β-1,4- and bonds. We the for during