Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum

BackgroundGalactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO.ResultsBoth GaO–CBM29 and CBM29–GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, k_cat/K_m values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO–CBM29.Major conclusionsSimilar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO–CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs.General significanceThis study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.     

Structural and thermodynamic dissection of specific mannan recognition by a carbohydrate binding module,TmCBM27

The C-terminal 176 amino acids of a Thermotoga maritima mannanase (Man5) constitute a carbohydrate binding module (CBM) that has been classified into CBM family 27. The isolated CBM27 domain, named TmCBM27, binds tightly (K(a)s 10(5)-10(6) M(-1)) to beta-1, 4-mannooligosaccharides, carob galactomannan, and konjac glucomannan, but not to cellulose (insoluble and soluble) or soluble birchwood xylan. The X-ray crystal structures of native TmCBM27, a TmCBM27-mannohexaose complex, and a TmCBM27-6(3),6(4)-alpha-D-galactosyl-mannopentaose complex at 2.0 A, 1.6 A, and 1.35 A, respectively, reveal the basis of TmCBM27's specificity for mannans. In particular, the latter complex, which is the first structure of a CBM in complex with a branched plant cell wall polysaccharide, illustrates how the architecture of the binding site can influence the recognition of naturally substituted polysaccharides.     

Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum

This study reports characteristics of different derivatives produced between CelA, a major endoglucanase of Clostridium thermocellum and carbohydrate binding domain of family 3a (CBM3a). In addition to the native form of the endoglucanase containing catalytic and dockerin domains (CelA-CD), its derivatives consisting of catalytic domain without dockerin domain (CelA-C), catalytic domain linked with the binding domain at N-, C- and both termini (CelA-BC, CelA-CB and CelA-BCB, respectively), two catalytic domains cloned in tandem (CelA-CC) and two catalytic domains intervened by a binding domain (CelA-CBC) were expressed in Escherichia coli at levels of 40, 43, 28, 30, 20, 20 and 10%, respectively of the total cell proteins. Specific activities of CelA-CD, CelA-C, CelA-BC, CelA-CB, CelA-CC, CelA-BCB and CelA-CBC against carboxymethyl cellulose (CMC) were 8.1, 7.0, 12.1, 8.5, 11.8, 10.2 and 23.5Umg(-1) enzyme while activities against pre-treated bagasse were 490, 250, 1400, 600, 810, 710 and 2270μmoles reducing sugars released per μmole of the enzyme, respectively, under the assay conditions used. Thus the activities of CelA-BC and CelA-CBC showed nearly 3- and 5-fold increase against pre-treated bagasse as compared to that of the native form of the enzyme, CelA-CD. Molecular modeling studies using MODELLER show that the binding residues of CBM3a and the active site residues of the catalytic domain are more favorably oriented for binding and hydrolysis of the polysaccharide in the case of CelA-BC as compared to those in CelA-CB, which corresponds with higher activity of the former.     

Evolution of a carbohydrate binding module into a protein-specific binder

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.     

Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

FmEG from Fomitiporia mediterranea is a non-modular endoglucanase composed of a 24-amino acids extension and 13-amino acids linker-like peptide at the N-terminus and a 312-amino acids GH5 catalytic domain (CD) at the C-terminus. In this study, six FmEG derivatives with deletion of N-terminal fragments or fusion with an extra family 1 carbohydrate-binding module (CBM1) was constructed in order to evaluate the contribution of CBM1 to FmEG processivity and catalytic activity. FmEG showed a weak processivity and released cellobiose (G2) and cellotriose (G3) as main end products, and cellotriose (G4) as minor end product from filter paper (FP), but more amount of G4 was released from regenerated amorphous cellulose (RAC). All derivatives had similar activity on carboxymethylcellulose (CMC) with the same optimal pH (7.0) and temperature (50 °C). However, fusing an extra CBM1 to FmEG△24 or FmEG△37 with flexible peptide significantly improved its processivity and catalytic activity to FP and RAC. Overall, 1.79- and 1.84-fold increases in the soluble/insoluble product ratio on FP, and 1.38- and 1.39-fold increases on RAC, compared to FmEG△24, were recorded for CBM1-FmEG△24 and CBM1-linker-FmEG△24, respectively. Meanwhile, they displayed 2.64- and 2.67-fold more activity on RAC, and 1.68- and 1.77-fold on FP, respectively. Similar improvement was also obtained for CBM1-linker-FmEG△37 as compared with FmEG△37. Interestingly, fusion of an extra CBM1 with FmEG also caused an alteration of cleavage pattern on insoluble celluloses. Our results suggest that such improvements in processivity and catalytic activity may arise from CBM1 binding affinity. The N-terminal 24- or 37-amino acids may serve as linker for sufficient spatial separation of the two domains required for processivity and catalytic activity. In addition, deletion of the N-terminal 24- or 37-amino acids led to significant reduction in thermostability but not the enzymatic activity.     

Analysis of glycoside hydrolase family 98: catalytic machinery, mechanism and a novel putative carbohydrate binding module

Glycoside hydrolases (GHs) are diverse enzymes of biotechnological and medical importance. Bioinformatics contributes to our understanding of GH structure and function in various ways, including dissection of their typically modular structures and detection of the distant evolutionary relationships between families that often allow for prediction of catalytic sites. Here these twin strands are applied to the recently described GH98 family, the founder member of which is a blood group glycotope-cleaving endo-beta-galactosidase of potential medical importance from Clostridium perfringens. Three domains can be discerned including a central catalytic TIM barrel domain in which putative catalytic residues can be assigned. Distant homologies and domain contexts suggest that the N-terminal domain is a novel carbohydrate binding module.     

Essential role of a family-32 carbohydrate-binding module in substrate recognition by Clostridium thermocellum mannanase CtMan5A

The family-5 glycoside hydrolase domain (GH5) and the family-32 carbohydrate-binding module (CBM32) of Clostridium thermocellum mannanase CtMan5A, along with their genetically inactivated derivatives, were collectively or separately expressed. Their catalytic and substrate-binding abilities were measured to investigate importance of CBM32 in substrate recognition by CtMan5A. Characterization of the truncated derivatives of CtMan5A and isothermal calorimetry analysis of the interaction between the inactivated proteins and mannooligosaccharides suggested that GH5 and CBM32 collectively formed a substrate-binding site capable of accommodating a mannotetraose unit in CtMan5A. This suggested that CBM32 directly participated in the substrate recognition required for catalytic action.     

Oligosaccharide recognition and binding to the carbohydrate binding module of AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) contains a carbohydrate-binding module (beta1-CBM) that is conserved from yeast to mammals. Beta1-CBM has been shown to localize AMPK to glycogen in intact cells and in vitro. Here we use Nuclear Magnetic Resonance spectroscopy to investigate oligosaccharide binding to 15N labelled beta1-CBM. We find that beta1-CBM shows greatest affinity to carbohydrates of greater than five glucose units joined via alpha,1-->4 glycosidic linkages with a single, but not multiple, glucose units in an alpha,1-->6 branch. The near identical chemical shift profile for all oligosaccharides whether cyclic or linear suggest a similar binding conformation and confirms the presence of a single carbohydrate-binding site.     

Genome-based identification of a carbohydrate binding module in Streptococcus pneumoniae hyaluronate lyase

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the connective tissues of higher animals, thereby destroying the normal connective tissue structure and exposing the host tissue cells to various endo- and exogenous factors, including bacterial toxins. The 3D crystal structures of functionally active but truncated Streptococcus pneumoniae and S. agalactiae hyaluronate lyases, along with their substrate and product complexes, have been determined. The enzymes are multidomain proteins with helical barrel-like catalytic domains and two types of beta-sheet domains. Here, through genome-based bioinformatics studies we identify an additional beta-sheet domain present in the most N-terminal part of streptococcal hyaluronate lyases. Fold recognition and modeling studies show that the domain is structurally similar to carbohydrate binding modules and is therefore likely to be directly involved in hyaluronan binding. Likely carbohydrate binding residues were identified and electrostatic complementarity of the hyaluronate lyase domain with hyaluronan demonstrated. The newly identified presumed hyaluronan binding domain likely improves catalytic efficiency by colocalizing the enzyme and its substrate. Other possible functions are discussed. Two contacting aromatic residues are conserved in the hydrophobic core of the hyaluronate lyase domain and in many, perhaps all, families in the superfamily in which they may be placed. This observation may help the identification and classification of other carbohydrate binding modules.     

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.     

The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.     

The crystal structure of the family 6 carbohydrate binding module from Cellvibrio mixtus endoglucanase 5a in complex with oligosaccharides reveals two distinct binding sites with different ligand specificities

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain non-catalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis. Family 6 CBMs (CBM6) are highly unusual because they contain two distinct clefts (cleft A and cleft B) that potentially can function as binding sites. Henshaw et al. (Henshaw, J., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes, C. M. G. A., and Gilbert, H. J. (2003) J. Biol. Chem. 279, 21552-21559) show that CmCBM6 contains two binding sites that display both similarities and differences in their ligand specificity. Here we report the crystal structure of CmCBM6 in complex with a variety of ligands that reveals the structural basis for the ligand specificity displayed by this protein. In cleft A the two faces of the terminal sugars of beta-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in CmCBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc-beta-1,4-Glc), subsite 4 will bind only to a beta-1,3-linked glucose, whereas subsite 1 can interact with either a beta-1,3- or beta-1,4-linked glucose. These different specificities of the subsites explain how cleft B can accommodate beta-1,4-beta-1,3- or beta-1,3-beta-1,4-linked gluco-configured ligands.     

1H, 13C and 15N backbone and side-chain resonance assignments of a family 36 carbohydrate binding module of xylanase from Paenibacillus campinasensis

Paenibacillus campinasensis BL11 isolated from black liquor secretes multiple glycoside hydrolases (GHs) against all kinds of polysaccharides. GH consists of a catalytic module and non-catalytic carbohydrate-binding modules (CBMs), in which CBMs append to the catalytic module, mediating specific interactions with insoluble carbohydrates to promote the hydrolysis efficiency of the cognate enzyme. Endo-β-1,4-xylanase (XylX) is one of the GHs reveals high enzymatic activity in a wide range of pH and thermal endurance, suitable for bioconversion and bio-refinement applications. In this work, we report the resonance assignments of a family 36 CBM (characterized as CBM36) derived from XylX. Our investigations will facilitate molecular structure determination and molecular dynamics analysis of CBMs.     

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.     

Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2

To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (α-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 °C that was 10 °C higher than the native Xyn2. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.     

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of β-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba.     

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5

Background

An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition.

A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K(d) of 200 microM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.     

Isothermal titration calorimetric studies on the binding of a family 6 carbohydrate-binding module of Clostridium thermocellum xynA with xlylooligosaccharides

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP = 2-8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5 x 10(3) M(-1) (DP = 2) to approximately 5 x 10(5) M(-1) (DP = 5-8) at 20 degrees C. The Ka values at 60 degrees C were about 1/10 of those at 20 degrees C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.