Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A.

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its alpha anomeric conformation and provide a rationale for specificity on p-nitrophenyl glycosides at the -1 and -2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth betaalpha-loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.     

Probing the structural basis for the difference in thermostability displayed by family 10 xylanases

Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution. Error-prone PCR and selection identified a double mutant, A334V/G348D, which confers an increase in thermostability. The mutant has a Tm 8 degrees C higher than the wild-type enzyme and, at 55 degrees C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 x 10(-4) min(-1), compared to a value of 1.6 x 10(-1) min(-1) for the wild-type enzyme. The introduction of the N to C-terminal disulphide bridge into A334V/G348D, which increases the thermostability of wild-type CmXyn10B, conferred a further approximately 2 degrees C increase in the Tm of the double mutant. The crystal structure of A334V/G348D showed that the introduction of Val334 fills a cavity within the hydrophobic core of the xylanase, increasing the number of van der Waals interactions with the surrounding aromatic residues, while O(delta1) of Asp348 makes an additional hydrogen bond with the amide of Gly344 and O(delta2) interacts with the arabinofuranose side-chain of the xylose moiety at the -2 subsite. To investigate the importance of xylan decorations in productive substrate binding, the activity of wild-type CmXyn10B, the mutant A334V/G348D, and several other GH10 xylanases against xylotriose and xylotriose containing an arabinofuranose side-chain (AX3) was assessed. The enzymes were more active against AX3 than xylotriose, providing evidence that the arabinose side-chain makes a generic contribution to substrate recognition by GH10 xylanases.     

Phototoxicity of protoporphyrin as related to its subcellular localization in mice livers after short-term feeding with griseofulvin.

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.     

The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition.

BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%. CONCLUSIONS: The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.     

A simple enzymatic synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a chromogenic substrate for assay and differentiation of endoxylanases

A simple procedure has been elaborated for preparation of 4-nitrophenyl beta-d-xylopyranosyl-1,4-beta-d-xylopyranoside (NPX(2)), a chromogenic substrate of some endo-beta-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl beta-d-xylopyranoside catalyzed by an Aureobasidium pullulans and Aspergillus niger beta-xylosidases. Both enzymes catalyzed only the formation of 4-nitrophenyl glycosides of beta-1,4-xylobiose with a small admixture of 4-nitrophenyl glycoside of beta-1,3-xylobiose. The highest yields of the NPX(2) (19.4%) was obtained at pH 5.5. The removal of the beta-1,3-isomer from NPX(2) is not necessary for quantification of endo-beta-1,4-xylanase activity since it is not attacked by endo-beta-1,4-xylanases. In contrast to GH family 5 xylanase from Erwinia chrysanthemi, which did not attack NPX(2), all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between xylobiose and the aromatic aglycone. Significant differences in the K(m) values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX(2). Moreover, NPX(2) could serve as an ideal substrate to follow the interaction of endo-beta-1,4-xylanases with various xylanase inhibitors.     

Xylanase and acetyl xylan esterase activities of XynA,a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation

The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.     

Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases

Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid. The xylanase from T. aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy. The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori. Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H. pylori.     

Novel Aspergillus hemicellulases enhance performance of commercial cellulases in lignocellulose hydrolysis

A novel hemicellulase-producing fungal strain was isolated from a local soil sample. The organism is identified as Aspergillus fumigatus based on ribosomal RNA analyses. The Aspergillus strain, designated as 2NB, produces both enzymes acting on xylan backbone (xylanase and β-xylosidase), and those acting on side chains (or accessory enzymes) notably α-arabinofuranosidase and acetyl-xylan esterase. The Asperigillus hemicellulases are characterized as having relatively low xylanase and β-xylosidase activities but high side chain removal activities. The activity ratio of side-chain acting enzymes to xylanase is higher than that of the Multifect enzyme, a commercial hemicellulase product. The potential of the novel hemicellulases in lignocelluloses bioprocessing was demonstrated with alkaline-pretreated switchgrass as lignocellulose substrate with hemicellulase supplemented with a ratio of xylanase activity to filter paper unit of 2:1. Supplement of Aspergillus hemicellulases to commercial cellulases significantly enhanced the hydrolysis of lignocellulose, achieving a 94% hydrolysis yield based on reducing sugar measurement, compared to 60% when no hemicellulase or 75% when Multifect enzyme was used under otherwise identical conditions. The significant improvement resulting from supplementing a hemicellulase mix with high side-chain removal activities suggests the importance of accessory hemicellulases in lignocellulose processing.     

Mango ripening--chemical and structural characterization of pectic and hemicellulosic polysaccharides

Ripening of mango is characterized by a gradual, but natural softening of the fruit, which is due to progressive depolymerization of pectic and hemicellulosic polysaccharides with significant loss of galactose, arabinose and mannose residues at the ripe stage. Structural characterization employing permethylation followed by GC-MS analysis, IR and ¹³C NMR measurements revealed the major CWS fractions of both unripe and ripe mangoes to be of variable molecular weights and having a 1,4-linked galactan/galacturonan backbone, which is occasionally involved in side chain branches consisting of single residues of galactose and arabinose or oligomeric 1,5-linked arabinofuranose residues linked through 1,3-linkages; whereas the major hemicellulosic fractions of unripe mango to be of xyloglucan-type having 1,4-linked glucan backbone with branching by non-reducing terminal arabinose and xylose residues.     

Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family

AXHs (arabinoxylan arabinofuranohydrolases) are alpha-L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a two-domain structure for this enzyme: a catalytic domain displaying a five-bladed beta-propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a beta-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, beta-xylosidase and alpha-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.     

Identification of new GH 10 and GH 11 xylanase genes from Aspergillus versicolor MKU3 by genome-walking PCR

Xylanases randomly clear the backbone of xylans, which are hemicelluloses representing a considerable source of fixed carbon in nature. Consequently, these enzymes have important industrial applications. To characterize the genes responsible for producing these enzymes, we cloned xylanase genes belonging to the GH11 and GH10 families from Aspergillus versicolor MKU3 using a 2-step polymerase chain reaction (PCR) protocol involving degenerate PCR and genome-walking PCR (GWPCR). We amplified a family 10 xylanase consensus fragment using degenerate PCR primers exhibiting specificity for conserved motifs within fungal family 10 xylanase genes. We identified a single family 10 xylanase gene (xynv10) and determined its entire gene sequence during the second step of GWPCR, which was used to amplify genomic DNA fragments upstream and downstream of xynv10. The xynv10 sequence contains a 1,378-bp open reading frame separated by 8 introns with an average size of 49 bp. We also amplified a partial GH11 xylanase gene sequence (xynv11) using degenerate PCR and genome-walking methods. Amplification of the C-terminal region of xynv11 using a degenerate primer designed from sequences revealed strong homology with the partial GH11 xylanase gene of A. versicolor MKU3. The structural region in xynv11 was approximately 680 bp and has one intron that is approximately 64 bp in length. Further expression and characterization of these genes will give better understanding of the role of these genes in xylan degradation by A. versicolor.     

Structural analysis of arabinoxylans isolated from native and malted finger millet (Eleusine coracana, ragi)

Structural elucidation of purified arabinoxylans isolated from finger millet and its malt by methylation, GLC-MS, periodate oxidation, Smith degradation, NMR, IR, optical rotation, and oligosaccharide analysis indicated that the backbone was a 1,4-beta-D-xylan, with the majority of the residues substituted at C-3. The major oligosaccharide generated by endo xylanase treatment was homogeneous with a molecular weight of 1865 Da corresponding to 14 pentose residues as determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural analysis of this oligosaccharide showed that it contained 8 xylose and 6 arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3 (disubstituted).     

Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thaliana isoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative "substrate specificity motif," only one residue, Met(293), was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met(293) or Lys(320), another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met(293), by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met(293) by bulky aromatic amino acids increased the apparent affinity (K(m)) for caffeate up to 10-fold, whereas single substitutions of Val(294) did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.     

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.     

A Bifunctional Endoglucanase/Endoxylanase from <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> with Potential Use in Industrial Processes at Different pH

Cellulomonas flavigena CDBB-531 was found to secrete a bifunctional cellulase/xylanase with a molecular mass of 49 kDa and pI 4.3. This enzyme was active on Remazol brilliant blue-carboxymethylcellulose (RBB-CMC) and Remazol brilliant blue-xylan (RBB-X). Based on thin-layer chromatographic analysis of the degradation products, the cellulase activity produced glucose, cellobiose, cellotriose, and cellotetraose from CMC as the substrate. When xylan from birchwood was used, end products were xylose, arabinose, and xylobiose. The bifunctional enzyme showed a pH optimum of 6 for cellulase activity and 9 for xylanase activity, which pointed out that this enzyme had separate sites for each activity. In both cases, the apparent optimum temperature was 50 degrees C. The predicted amino acid sequence of purified protein showed similarity with the catalytic domain of several glycosyl hydrolases of family 10.     

High resolution structure and sequence of T. aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture.

Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins. Proteins 1999;36:295-306.     

Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding

Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus beta-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical beta-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two beta-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.