Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with alpha-l-arabinofuranosyl- and 4-O-methyl-alpha-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains alpha and gamma of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper, demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.     

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

Branching enzymes (BEs) catalyze the formation of branch points in glycogen and amylopectin by cleavage of α-1,4 glycosidic bonds and subsequent transfer to a new α-1,6 position. BEs generally belong to glycoside hydrolase family 13 (GH13); however TK1436, isolated from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, is the first GH57 member, which possesses BE activity. To date, the only BE structure that had been determined is a GH13-type from Escherichia coli. Herein, we have determined the crystal structure of TK1436 in the native state and in complex with glucose and substrate mimetics that permitted mapping of the substrate-binding channel and identification of key residues for glucanotransferase activity. Its structure encompasses a distorted (β/α)(7)-barrel juxtaposed to a C-terminal α-helical domain, which also participates in the formation of the active-site cleft. The active site comprises two acidic catalytic residues (Glu183 and Asp354), the polarizer His10, aromatic gate-keepers (Trp28, Trp270, Trp407, and Trp416) and the residue Tyr233, which is fully conserved among GH13- and GH57-type BEs. Despite TK1436 displaying a completely different fold and domain organization when compared to E. coli BE, they share the same structural determinants for BE activity. Structural comparison with AmyC, a GH57 α-amylase devoid of BE activity, revealed that the catalytic loop involved in substrate recognition and binding, is shortened in AmyC structure and it has been addressed as a key feature for its inability for glucanotransferase activity. The oligomerization has also been pointed out as a possible determinant for functional differentiation among GH57 members.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Structure and function of a family 10 beta-xylanase chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.     

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.     

Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis

Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6A resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (betaalpha)(8)-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5.     

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.     

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs

Background

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.

Results

Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism.

Conclusions

The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.     

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-β-D-mannanase

The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.     

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Endo-alpha-1-4-polygalactosaminidases and their homologues: structure and evolution

Endo-alpha-1,4-polygalactosaminidase is a rare enzyme. Its catalytic domain belongs to the GH114 family of glycoside hydrolases. Phylogenetic analysis of the family proteins allowed us to show an important role of duplications, eliminations, and horizontal transfer in the evolution of their genes. Domain structure, the secondary structure, and proposed structure of the active center of the endo-alpha-1,4-polygalactosaminidases are discussed. Evolutionary connections of the GH114 family with GH13, GH18, GH20, GH27, GH29, GH31, GH35, GH36, and GH66 families of glycoside hydrolases, as well as, with COG1306, COG1649, COG2342, GHL3, and GHL4 families of enzymatically uncharacterized proteins have been revealed by iterative screening of the protein database. The unclassified homologues have been grouped into 13 new families of hypothetical glycoside hydrolases: GHL5 - GHL15, GH36J, and GH36K.     

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.     

Structural analyses and yeast production of the β-1,3-1,4-glucanase catalytic module encoded by the licB gene of Clostridium thermocellum

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris, encouraging its further commercial applications.     

Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H_f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.     

Endo-α-1,4-polygalactosaminidases and their homologs: Structure and evolution

Endo-α-1,4-polygalactosaminidase is a rare enzyme. Its catalytic domain belongs to the GH114 family of glycoside hydrolases. It is shown by phylogenetic analysis that the evolution of the corresponding genes involved duplications, elimination, and horizontal transfer. The domain and secondary structures of endo-α-1,4-polygalactosaminidases are discussed. A hypothesis is put forward as to the structure of the active center of the enzyme. Iterative screening of a protein database reveals evolutionary relationships of the GH114 family with the GH13, GH18, GH20, GH27, GH29, GH31, GH35, GH36, and GH66 families of glycoside hydrolases and with the COG1306, COG1649, COG2342, GHL3, and GHL4 families of proteins with unknown enzymatic functions. Unclassified homologs are grouped into 13 new families of hypothetical glycoside hydrolases: GHL5-GHL15, GH36J, and GH36K.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the −1 subsite or prefer xylosyl-substituted residues in the −1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

     

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site

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 beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-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 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), 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 beta-1,4- and beta-1,3-1,4-mixed linked glucans.     

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Å resolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite −1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.