Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.     

X-ray crystal structure of a non-crystalline cellulose-specific carbohydrate-binding module: CBM28

Natural cellulose exists as a composite of different forms, which have historically been broadly characterized as "crystalline" or "amorphous". The recognition of both of these forms of cellulose by the carbohydrate-binding modules (CBM) of microbial glycoside hydrolases is central to natural and efficient biotechnological conversion of plant cell wall biomass. There is increasing evidence that, at least some, individual binding modules target distinct and different regions of non-crystalline "amorphous" cellulose. Competition experiments show that CBM28 modules do not compete with CBM17 modules when binding to non-crystalline cellulose. The structure of the BspCBM28 (http://afmb.cnrs-mrs.fr/CAZY/) module from the Bacillus sp. 1139 family GH5 endoglucanase, comprising a 191 amino acid protein, has therefore been determined at 1.4A resolution using single isomorphous replacement with anomalous scattering methods. The structure reveals a "beta-jelly roll" topology, with high degree of similarity to the structure of CBM17 domains. Sequence and structural conservation strongly suggests that these two families of domains have evolved through gene duplication and subsequent divergence. The ligand-binding site "topographies" of CBMs from families 28, 17 and 4 begins to shed light on the differential recognition of non-crystalline cellulose by multi-modular plant cell wall-degrading enzymes.     

Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a xylanase that contains a family 3 carbohydrate-binding module

Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.     

Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module

The crystal structures of a carbohydrate-binding module (CBM) family 28 domain of endoglucanase Cel5A from Clostridium josui have been determined in ligand-free and complex forms with cellobiose, cellotetraose, and cellopentaose as the first complex structures of this family. In the cleft of a β-sandwich fold, the ligands are recognized by stacking interactions and hydrogen bonds. Conformations of the bound cellooligosaccharides are similar to those in crystals and solution but clearly different from the cellulose structure. Interestingly, the glucan chain bound on CBM28 is in the opposite direction of that bound to CBM17, although these families share significant structural similarity.     

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.     

Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis

Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.     

Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.     

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

The three-dimensional model of the CtCBM35 (Cthe 2811), i.e. the family 35 carbohydrate binding module (CBM) from the Clostridium thermocellum family 26 glycoside hydrolase (GH) β-mannanase, generated by Modeller9v8 displayed predominance of β-sheets arranged as β-sandwich fold. Multiple sequence alignment of CtCBM35 with other CBM35s showed a conserved signature sequence motif Trp-Gly-Tyr, which is probably a specific determinant for mannan binding. Cloned CtCBM35 from Clostridium thermocellum ATCC 27405 was a homogenous, soluble 16 kDa protein. Ligand binding analysis of CtCBM35 by affinity electrophoresis displayed higher binding affinity against konjac glucomannan (K _a = 2.5 × 10⁵ M^?1) than carob galactomannan (K _a = 1.4 × 10⁵ M^?1). The presence of Ca²⁺ ions imparted slightly higher binding affinity of CtCBM35 against carob galactomannan and konjac glucomannan than without Ca²⁺ ion additive. However, CtCBM35 exhibited a low ligand-binding affinity K _a = 2.5 × 10^?5 M^?1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed K _a against konjac glucomannan and carob galactomannan, 2.4 × 10⁵ M^?1 and 1.44 × 10⁵ M^?1, and ΔG of binding ?27.0 and ?25.0 kJ/mol, respectively, substantiating the findings of affinity electrophoresis. Ca²⁺ ions escalated the thermostability of CtCBM35 and its melting temperature was shifted to 70°C from initial 55°C. Therefore thermostable CtCBM35 targets more β-(1,4)-manno-configured ligands from plant cell wall hemicellulosic reservoir. Thus a non-catalytic CtCBM35 of multienzyme cellulosomal enzymes may gain interest in the biofuel and food industry in the form of released sugars by targeting plant cell wall polysaccharides.     

Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.     

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.     

Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module

Objective

To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA.

Results

Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K _m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T₅₀ and a 2.9 °C higher T_m than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content.

Conclusions

The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.

     

Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.     

Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A

Plant cell wall hydrolases generally have a modular structure consisting of a catalytic domain linked to one or more noncatalytic carbohydrate-binding modules (CBMs), whose common function is to attach the enzyme to the polymeric substrate. Xylanase A from Pseudomonas fluorescens subsp. cellulosa (Pf Xyn10A) consists of a family 10 catalytic domain, an N-terminal family IIa cellulose-binding module, and an internal family 10 cellulose-binding module. The structure of the 45-residue family 10 CBM has been determined in solution using NMR. It consists of two antiparallel beta-sheets, one with two strands and one with three, with a short alpha-helix across one face of the three-stranded sheet. There is a high density of aromatic residues on one side of the protein, including three aromatic residues (Tyr8, Trp22, and Trp24), which are exposed and form a flat surface on one face, in a classical polysaccharide-binding arrangement. The fold is closely similar to that of the oligonucleotide/oligosaccharide-binding (OB) fold, but appears to have arisen by convergent evolution, because there is no sequence similarity, and the presumed binding sites are on different faces.     

Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module

We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M _r) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.     

The first crystal structure of a family 31 carbohydrate-binding module with affinity to beta-1,3-xylan

Here, we present the crystal structure of the family 31 carbohydrate-binding module (CBM) of beta-1,3-xylanase from Alcaligenes sp. strain XY-234 (AlcCBM31) determined at a resolution of 1.25A. The AlcCBM31 shows affinity with only beta-1,3-xylan. The AlcCBM31 molecule makes a beta-sandwich structure composed of eight beta-strands with a typical immunoglobulin fold and contains two intra-molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight beta-strands comprise a beta-sandwich structure with a typical jelly-roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.     

Engineered xyloglucan specificity in a carbohydrate-binding module

The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.     

Molecular engineering of a thermostable carbohydrate-binding module

Structure–function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel?, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

Molecular engineering of a thermostable carbohydrate-binding module

Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel™, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.