The structural basis for the ligand specificity of family 2 carbohydrate-binding modules

The interactions of proteins with polysaccharides play a key role in the microbial hydrolysis of cellulose and xylan, the most abundant organic molecules in the biosphere, and are thus pivotal to the recycling of photosynthetically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). The largest prokaryotic CBM family, CBM2, contains members that bind cellulose (CBM2a) and xylan (CBM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate interaction is rotated by 90 degrees compared with its position in CBM2a (thus matching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Arg-262-->Gly), a CBM2b xylan-binding module completely loses its affinity for xylan and becomes a cellulose-binding module. The structural effect of the mutation has been revealed using NMR spectroscopy, which confirms that Trp-259 rotates 90 degrees to lie flat against the protein surface. Except for this one residue, the mutation only results in minor changes to the structure. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the recognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.     

Alternative splicing produces two endoglucanases with one or two carbohydrate-binding modules in Mucor circinelloides

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, that were isolated from Rhizopus oryzae as the first cellulase genes from a member of the subdivision Zygomycota. In this study, two cDNAs homologous to the rce1 gene, designated the mce1 and mce2 cDNAs, were cloned from Mucor circinelloides, a member of the subdivision Zygomycota. The mce1 cDNA encoded an endoglucanase (family 45 glycoside hydrolase) having one carbohydrate-binding module (CBM), designated MCE1, and the mce2 cDNA encoded the same endoglucanase having two tandem repeated CBMs, designated MCE2. The two cDNAs contained the same sequences but with a 147-bp insertion. The corresponding genomic mce gene consisted of four exons. The mce1 cDNA was created from exons 1, 3, and 4, and the mce2 cDNA was created from exons 1, 2, 3, and 4. These results indicate that the mce1 and mce2 cDNAs were created from one genomic mce gene by alternative splicing. MCE1 and MCE2, purified to apparent homogeneity from the culture supernatant of M. circinelloides, had molecular masses of 43 and 47 kDa, respectively. The carboxymethyl cellulase specific activity of MCE2 was almost the same as that of MCE1, whereas the Avicelase specific activity of MCE2 was two times higher than that of MCE1. Furthermore, MCE2, whose two tandem CBMs might be more effective for degradation of crystalline cellulose than one CBM, was secreted only at an early culture stage when crystalline cellulose was abundant.     

CBM在多糖底物降解中的应用进展

碳水化合物结合模块(carbohydrate-binding module, CBM)是碳水化合物活性酶的重要组成部分,其功能是识别并结合到特定的多糖底物上以提高催化结构域在底物附近的浓度及催化效率,帮助其更好地降解如纤维素、木聚糖、几丁质和黄原胶等大分子化合物。不同家族的CBM因其来源或结构不同往往会具有不同的底物结合特性。本文从CBM的家族、结构和功能等方面对CBM近年来的研究进行了综述,特别是对其作为融合单元运用到多糖底物的降解和糖苷水解酶改造方面的应用进行了总结。     

Molecular basis for the selectivity and specificity of ligand recognition by the family 16 carbohydrate-binding modules from Thermoanaerobacterium polysaccharolyticum ManA

Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward beta-1,4-linked sugars, they can engage both cello- and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.     

X4 modules represent a new family of carbohydrate-binding modules that display novel properties

The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.     

Co-operative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase

Family 6 carbohydrate-binding modules were amplified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from approximately 3 x 10(3) M(-1) to approximately 1 x 10(5) M(-1). Tandem and triplet combinations of these modules bound co-operatively to soluble xylan and insoluble cellulose to give approximately 20- to approximately 40-fold increases in affinity relative to the individual modules. This co-operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simultaneously with proximal binding sites on the polysaccharides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co-operativity in these multiplets may compensate for the loss of affinity at higher temperatures.     

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.     

Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.     

A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins

We recently showed that some of the enzymes underpinning cellulose solubilization by Ruminococcus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an "X" domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus.     

Insight into ligand diversity and novel biological roles for family 32 carbohydrate-binding modules

Family 32 carbohydrate-binding modules (CBM32s) are found in a diverse group of microorganisms, including archea, eubacteria, and fungi. Significantly, many members of this family belong to plant and animal pathogens where they are likely to play a key role in enzyme toxin targeting and function. Indeed, ligand targets have been shown to range from insoluble plant cell wall polysaccharides to complex eukaryotic glycans. Besides a potential direct involvement in microbial pathogenesis, CBM32s also represent an important family for the study of CBM evolution due to the wide variety of complex protein architectures that they are associated with. This complexity ranges from independent lectin-like proteins through to large multimodular enzyme toxins where they can be present in multiple copies (multimodularity). Presented here is a rigorous analysis of the evolutionary relationships between available polypeptide sequences for family 32 CBMs within the carbohydrate active enzyme database. This approach is especially helpful for determining the roles of CBM32s that are present in multiple copies within an enzyme as each module tends to cluster into groups that are associated with distinct enzyme classes. For enzymes that contain multiple copies of CBM32s, however, there are differential clustering patterns as modules can either cluster together or in very distant sections of the tree. These data suggest that enzymes containing multiple copies possess complex mechanisms of ligand recognition. By applying this well-developed approach to the specific analysis of CBM relatedness, we have generated here a new platform for the prediction of CBM binding specificity and highlight significant new targets for biochemical and structural characterization.     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk_on) and desorption (k_off) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.     

Affinity electrophoresis for the identification and characterization of soluble sugar binding by carbohydrate-binding modules

Affinity electrophoresis was used to identify and quantify the interaction of carbohydrate-binding modules (CBMs) with soluble polysaccharides. Association constants determined by AE were in excellent agreement with values obtained by isothermal titration calorimetry and fluorescence titration. The method was adapted to the identification, study and characterization of mutant carbohydrate-binding modules with altered affinities and specificities. Competition affinity electrophoresis was used to monitor binding of small, soluble mono- and disaccharides to one of the modules.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue¹⁹². Site-directed mutagenesis of three active site residues of meso-BDH, including Trp¹⁹⁰, which corresponds to Trp¹⁹² of l-BDH, converted its stereospecificity to that of l-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.     

Divergent modes of glycan recognition by a new family of carbohydrate-binding modules

The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.     

Functions of family-22 carbohydrate-binding modules in Clostridium josui Xyn10A

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.     

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers

Novel molecular probes have been developed for the analysis and detection of polysaccharides in plant cell walls using carbohydrate-binding modules (CBMs) derived from modular glycoside hydrolases belonging to families 2a, 6, and 29. Recombinant forms of these proteins containing his-tags, in conjunction with anti-his-tag detection, provide a flexible system that utilizes CBMs as molecular probes in a range of applications. Assays for the rapid analysis of the binding of CBMs to polysaccharides and oligosaccharides using nitrocellulose-based CBM macroarrays and microtiter plate-based CBM capture and competitive-inhibition assays are described. We also demonstrate the use of CBMs with his-tags for the localization of their target ligands in planta. The generation of molecular probes from other families of CBMs will dramatically increase the repertoire of molecular probes available to determine the developmental and functional aspects of plant cell walls.