De novo prediction of three-dimensional structures for major protein families

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.     

High-resolution crystal structures of the lectin-like xylan binding domain from Streptomyces lividans xylanase 10A with bound substrates reveal a novel mode of xylan binding

Carbohydrate-binding module (CBM) family 13 includes the "R-type" or "ricin superfamily" beta-trefoil lectins. The C-terminal CBM, CBM13, of xylanase 10A from Streptomyces lividans is a family 13 CBM that is not only structurally similar to the "R-type" lectins but also somewhat functionally similar. The primary function of CBM13 is to bind the polysaccharide xylan, but it retains the ability of the R-type lectins to bind small sugars such as lactose and galactose. The association of CBM13 with xylan appears to involve cooperative and additive participation of three binding pockets in each of the three trefoil domains of CBM13, suggesting a novel mechanism of CBM-xylan interaction. Thus, the interaction of CBM13 with sugars displays considerable plasticity for which we provide a structural rationale. The high-resolution crystal structure of CBM13 was determined by multiple anomalous dispersion from a complex of CBM13 with a brominated ligand. Crystal structures of CBM13 in complex with lactose and xylopentaose revealed two distinct mechanisms of ligand binding. CBM13 has retained its specificity for lactose via Ricin-like binding in all of the three classic trefoil binding pockets. However, CBM13 has the ability to bind either the nonreducing galactosyl moiety or the reducing glucosyl moiety of lactose. The mode of xylopentaose binding suggests adaptive mutations in the trefoil sugar binding scaffold to accommodate internal binding on helical polymers of xylose.     

Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites

The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units. The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide. Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition. The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1. Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding. In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive. This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.     

Purification and characterization of a carbohydrate: acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently

A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized beta-(1-->4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the beta-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.     

Purification and some properties of five endo-1,4-beta-D-xylanases and a beta-D-xylosidase produced by a strain of Aspergillus niger.

Five different xylanases and a beta-D-xylosidase in the culture medium of Aspergillus niger have been purified to homogeneity from 13- to 52-fold by a procedure of gel and hydroxylapatite chromatography. The strain was isolated from soil of the African equatorial forest. Gel chromatography of the purified enzymes indicated that three of the xylanases have molecular weights of 31,000 and the other two xylanases have molecular weights of 50,000. beta-D-Xylosidase has a molecular weight of 78,000. The pH curves of the xylanases were quite diverse and showed pH optima ranging from 4.0 to 6.5. Characteristic action patterns were obtained for each of the purified xylanases by gel chromatography of the xylan digests on Bio-Gel P-2. The enzymes degraded arabinoxylan by an endomechanism, producing L-arabinose, D-xylose, xylobiose, and a mixture of branched arabinose-xylose and D-xylose oligosaccharides. All xylanases seemed to be capable of liberating L-arabinose from either arabinoxylan or the arabinose-xylose oligosaccharides. Branched arabinose-containing D-xylose oligosaccharides were slowly hydrolyzed, so that these sugars accumulate in the digest. Two xylanases showed relatively broad substrate specificity and were able to degrade also crystalline cellulose. beta-D-Xylosidase showed optimal activity at pH 6.7 to 7.0 and at 42 degrees C. The Km for o-nitrophenyl-beta-D-xylopyranoside was 0.22 mM and xylotriose was hydrolyzed more rapidly than xylobiose.     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K_m and V_max values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.     

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.     

Simultaneous catalysis and product separation by cross-linked enzyme crystals

Crystalline cross-linked xylose isomerase (CLXI, EC 5.3.1.5) and xylanase (CLX, EC 3.2.1.8) were studied in a packed-bed reactor for simultaneous catalytic reaction and separation of substrates from reaction products. Streptomyces rubiginosus xylose isomerase catalyzed a slow isomerization of L-arabinose to L-ribulose and an epimerization to L-ribose. In equilibrium the reaction mixture contained 52.5% arabinose, 22.5% ribulose, and 25% ribose. In a packed-bed column filled with CLXI, a simultaneous reaction and separation resulted in fractions where arabinose concentration varied between 100-0%, ribulose between 0-55%, and ribose between 0-100%. Trichoderma reesei xylanase II hydrolyzed and transferred xylotetraose mainly to xylotriose and xylobiose. In a packed-bed column filled with CLX, xylotetraose rapidly reacted to xylobiose and xylose by a mechanism that is not yet fully understood.     

Conversion of sugars to 1,2-propanediol by Thermoanaerobacterium thermosaccharolyticum HG-8

The purpose of this study was to explore a fermentation route for the production of 1,2-propanediol (1,2-PD) from renewable sugars: lactose found in cheese whey, and D-glucose, D-galactose, L-arabinose, and D-xylose found in corn and wood byproducts. Thermoanaerobacterium thermosaccharolyticum, a naturally occurring organism, was found to ferment a wider range of sugars to 1,2-PD than previously reported. The specific sugar had a significant effect on the selectivity for 1,2-PD vs other fermentation products such as ethanol, D- and L-lactate, and acetate. T. thermosaccharolyticum potentially provides an environmentally friendly route to a major commodity chemical now made from petrochemicals.     

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae

Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.     

Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.     

Rapid analysis of mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass by HPLC

HPLC using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to analyse mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass. PMP derivatives, including those of mannose, rhamnose, cellobiose, glucose, xylose and arabinose, were separated within 14 min with detection at 254 nm. The method was also suitable for xylo-oligosaccharides (XOS): PMP derivatives of xylohexaose, xylopentaose, xylotetraose, xylotriose and xylobiose were well separated under the same conditions. The method was used to determine the mono-saccharide composition of Miscanthus and evaluate the production of XOS from enzymatic hydrolysis of crude xylan.     

Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.     

Carbon-13 nuclear magnetic resonance study of microtubule protein: evidence for a second colchicine site involved in the inhibition of microtubule assembly

A 13C nuclear magnetic resonance study of bovine microtubule protein was carried out at 43 kG in the presence and absence of colchicine 13C labeled at the tropolone methoxy. Analysis indicated that tubulin has at least two colchicine binding sites: a quasi-irreversibly bound, high-affinity site (i.e., the KD less than 5 microM site generally accepted as the site of colchicine action) as well as a low-affinity site(s) (KD approximately 650 microM) with which free colchicine rapidly exchanges (greater than 100 s-1). The methoxy resonance is broadened to different apparent extents as a result of binding at these two sites (50- vs. 150-Hz broadening for the high- and low-affinity sites, respectively) but undergoes no change in chemical shift upon binding. The low-affinity sites are interpreted to be analogous to the sites deduced by Schmitt and Atlas [Schmitt, H., & Atlas, D. (1976) J. Mol. Biol. 102, 743-758] from labeling studies using bromocolchicine. These sites are likely to be the sites responsible for the abrupt halt in microtubule assembly ("capping") observed at high colchicine concentrations (greater than 20 microM)--a qualitatively different behavior from that observed at low colchicine concentrations [Sternlicht, H., Ringel, I., & Szasz, J. (1983) Biophys. J. 42, 255-267]. Carbon-13 spectra from the aliphatic carbons of microtubule protein consists of narrow resonances--many with line widths less than 30 Hz--superimposed on a broad background. The narrow resonances were assigned to flexible regions in nontubulin proteins [microtubule-associated proteins (MAPs)], in accord with an earlier 1H nuclear magnetic resonance study of microtubule protein [Woody, R. W., Clark, D. C., Roberts, G. C. K., Martin, S. R., & Bayley, P. M. (1983) Biochemistry 22, 2186-2192]. This assignment was supported by 13C NMR analysis of phosphocellulose-purified (MAP-depleted) tubulin as well as heat-stable MAPs. Aliphatic carbons in the MAP preparations were characterized by narrow resonances indicative of carbons with considerable motional freedom whereas the aliphatic regions of phosphocellulose-purified tubulin were, for the most part, characterized by broad resonances indicative of carbons with restricted mobility. However, a moderately narrow resonance (approximately less than 50-Hz line width) coincident with the C gamma resonance of glutamate was detected in 13C NMR spectra of tubulin which indicated that a fraction of the glutamic acid residues is relatively mobile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

Cellobiose chemotaxis by the cellulolytic bacterium Cellulomonas gelida.

In the course of a study on the bacterial degradation of plant cell wall polysaccharides, we observed that growing cells of motile cellulolytic bacteria accumulated, without attachment, near cellulose fibers present in the cultures. Because it seemed likely that the accumulation was due to chemotactic behavior, we investigated the chemotactic responses of one of the above-mentioned bacteria (Cellulomonas gelida ATCC 488). We studied primarily the responses toward cellobiose, which is the major product of cellulose hydrolysis by microorganisms, and toward hemicellulose hydrolysis products. We found that cellobiose, cellotriose, D-glucose, xylobiose, and D-xylose, as well as other sugars that are hemicellulose components, served as chemoattractants for C. gelida, as determined by a modification of Adler's capillary assay. Competition and inducibility experiments indicated that C. gelida possesses at least two types of separately regulated cellobiose chemoreceptors (Cb1 and cellobiose, cellotriose, xylobiose, and D-glucose, and it is constitutively synthesized. The presence in C. gelida of a constitutive response toward cellobiose and of at least two distinct cellobiose chemoreceptors has implications for the survival of this cellulolytic bacterium in nature. A possible mechanism for cellobiose-mediated bacterial chemotaxis toward cellulose is proposed. We suggest that, in natural environments, motile cellulolytic bacteria migrate toward plant materials that contain cellulose and hemicellulose by swimming up cellobiose concentration gradients and/or concentration gradients of other sugars (e.g., xylobiose, D-xylose, and D-glucose) formed by enzymatic hydrolysis of plant cell wall polysaccharides.     

Crystal structures of the family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A in native and ligand-bound forms

The C-terminal module of the thermostable Thermotoga maritima xylanase 10A (CBM9-2) is a family 9 carbohydrate-binding module that binds to amorphous and crystalline cellulose and a range of soluble di- and monosaccharides as well as to cello and xylo oligomers of different degrees of polymerization [Boraston, A. B., Creagh, A. L., Alam, Md. M., Kormos, J. M., Tomme, P., Haynes, C. A., Warren, R. A. J., and Kilburn, D. G. (2001) Biochemistry 40, 6240-6247]. The crystal structure of CBM9-2 has been determined by the multiwavelength anomalous dispersion method to 1.9 A resolution. CBM9-2 assumes a beta-sandwich fold and contains three metal binding sites. The bound metal atoms, which are most likely calcium cations, are in an octahedral coordination. The crystal structures of CBM9-2 in complex with glucose and cellobiose were also determined in order to identify the sugar-binding site and provide insight into the structural basis for sugar binding by CBM9-2. The sugar-binding site is a solvent-exposed slot sufficient in depth, width, and length to accommodate a disaccharide. Two tryptophan residues are stacked together on the surface of the protein forming the sugar-binding site. From the complex structures with glucose and cellobiose, it was inferred that CBM9-2 binds exclusively to the reducing end of mono-, di-, and oligosaccharides with an intricate hydrogen-bonding network involving mainly charged residues, as well as stacking interactions by Trp175 and Trp71. The binding interactions are limited to disaccharides as was expected from calorimetric data. Comparison of the glucose and cellobiose complexes revealed surprising differences in binding of these two substrates by CBM9-2. Cellobiose was found to bind in a distinct orientation from glucose, while still maintaining optimal stacking and electrostatic interactions with the reducing end sugar.     

The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain

Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose. Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A. The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules.