Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding

The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates. These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst. His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides.     

Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.     

The active site of human glucocerebrosidase: structural predictions and experimental validations

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase which cleaves the beta-glucosidic linkage of glucosylceramide, a normal intermediate in glycolipid metabolism. Glucocerebrosidase belongs to the clan GH-A of glycoside hydrolases, a large group of enzymes which function with retention of the anomeric configuration at the hydrolysis site. Accurate three-dimensional (3D) structure data for glucocerebrosidase should help to better understand the molecular bases of Gaucher disease. As such 3D structure data were not available, we used the two-dimensional hydrophobic cluster analysis (HCA) method to make structure predictions for the catalytic domains of clan GH-A glycoside hydrolases. We found that all the enzymes of clan GH-A may share a similar catalytic domain consisting of an (alpha/beta)8 barrel with the critical acid/base and nucleophile residues located at the C-terminal ends of strands beta 4 and beta 7, respectively. In the case of glucocerebrosidase, Glu 235 was predicted to be the putative acid/base catalyst whereas the nucleophile was located at Glu 340. Next, in order to obtain experimental evidence supporting these HCA-based predictions, we used retroviral vectors to express, in murine null cells, E235A and E340A mutant proteins, in which alanine residues unable to participate in the enzymatic reaction replace the presumed critical glutamic acid residues. Both mutants were found to be catalytically inactive although they were correctly folded/processed and sorted to the lysosome. Thus, Glu 235 and Glu 340 do indeed play key roles in the active site of human glucocerebrosidase as predicted by the HCA analysis. In a broader perspective, our work points out that bioinformatics approaches may be highly useful for generating structure-function predictions based on sequence-structure interrelationships, especially in the context of a rapid increase in protein sequence information through genome sequencing.     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A

The three-dimensional structure of Aspergillus aculeatus beta-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (beta/alpha)(8) barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate-enzyme interactions were elucidated by modeling of beta-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A comparison with the substrate binding grooves of other Clan GH-A enzymes suggests that shape complementarity is crucial in determining the specificity of polysaccharidases.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Identification of active-site residues of the pro-metastatic endoglycosidase heparanase

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).     

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.     

Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.     

Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 A. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a beta-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu(186) and Glu(359), are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a (1)H NMR experiment using the reduced cellooligosaccharide as a substrate.     

Structural and functional analysis of a glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron

SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6A resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu532 is positioned to provide protonic assistance to leaving group departure, with Glu439 and Glu508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.     

Function and structure studies of GH family 31 and 97 α-glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

The structural basis of substrate recognition in an exo-beta-D-glucosaminidase involved in chitosan hydrolysis

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with β-galactosidase activity (Escherichia coli LacZ), β-glucuronidase activity (Homo sapiens GusB), and β-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-β-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 Å resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural β-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-β-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.     

The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu²²⁷ and Glu²⁶⁸, hydrogen bond to O₁ of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys¹⁹⁹ in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages.     

Structural analysis of CPF_2247, a novel α-amylase from Clostridium perfringens

CPF_2247 from Clostridium perfringens ATCC 13124 was identified as a putative carbohydrate‐active enzyme by its low sequence identity to endo‐β‐1,4‐glucanases belonging to family 8 of the glycoside hydrolase classification. The X‐ray crystal structure of CPF_2247 determined to 2.0 Å resolution by single‐wavelength anomalous dispersion using seleno‐methionine‐substituted protein revealed an (α/α)₆ barrel fold. A large cleft on the surface of the protein contains residues that are structurally conserved with key elements of the catalytic machinery in clan GH‐M glycoside hydrolases. Assessment of CPF_2247 as a carbohydrate‐active enzyme disclosed α‐glucanase activity on amylose, glycogen, and malto‐oligosaccharides. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Investigating the binding of β‐1,4‐galactan to Bacillus licheniformis β‐1,4‐galactanase by crystallography and computational modeling

Microbial β‐1,4‐galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH‐A of glycoside hydrolases, which cover many different poly‐ and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis β‐1,4‐galactanase and its inactive nucleophile mutant have been obtained with methyl‐β(1→4)‐galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the β‐1,4‐galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a β(1→4)‐galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite ?4 to +5. In particular, this analysis newly identified a conserved β‐turn, which contributes to subsites ?2 to +3. This β‐turn is unique to family 53 β‐1,4‐galactanases among all clan GH‐A families that have been structurally characterized and thus might be a structural signature for endo‐β‐1,4‐galactanase specificity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Crystallographic analysis shows substrate binding at the -3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-beta-xylanases

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.     

Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum

Numerous bacterial and fungal organisms have evolved elaborate sets of modular glycoside hydrolases and similar enzymes aimed at the degradation of polymeric carbohydrates. Presently, on the basis of sequence similarity catalytic modules of these enzymes have been classified into 90 families. Representatives of a particular family display similar fold and catalytic mechanisms. However, within families distinctions occur with regard to enzymatic properties and type of activity against carbohydrate chains. Cellobiohydrolase CbhA from Clostridium thermocellum is a large seven-modular enzyme with a catalytic module belonging to family 9. In contrast to other representatives of that family possessing only endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an exocellulase. The crystal structures of the combination of the immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT) are reported here. The detailed analysis of these structures reveals that, while key catalytic residues and overall fold are conserved in this enzyme and those of other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off after the -2 subsite. This feature which is created by an extension and altered conformation of a single loop region explains the inability of the active site of CbhA to accommodate a long cellulose chain and to cut it internally. This altered loop region is responsible for the exocellulolytic activity of the enzyme.