Carbohydrate-binding Module Family Research Articles

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians.

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

Carbohydrate-binding module tribes.

At present, 69 families of carbohydrate-binding modules (CBMs) have been isolated by statistically significant differences in the amino acid sequences (primary structures) of their members, with most members of different families showing little if any homology. On the other hand, members of the same family have primary and tertiary (three-dimensional) structures that can be computationally aligned, suggesting that they are descended from common protein ancestors. Members of the large majority of CBM families are β-sandwiches. This raises the question of whether members of different families are descended from distant common ancestors, and therefore are members of the same tribe. We have attacked this problem by attempting to computationally superimpose tertiary structure representatives of each of the 53 CBM families that have members with known tertiary structures. When successful, we have aligned locations of secondary structure elements and determined root mean square deviations and percentages of similarity between adjacent amino acid residues in structures from similar families. Further criteria leading to tribal membership are amino acid chain lengths and bound ligands. These considerations have led us to assign 27 families to nine tribes. Eight of the tribes have members with β-sandwich structures, while the ninth is composed of structures with β-trefoils.

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw.

BackgroundNew lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes.ResultsDifferential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora.ConclusionsCoupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis.

BackgroundBacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7.ResultsA comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon.ConclusionsOur data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1066) contains supplementary material, which is available to authorized users.

Two structurally related starch-binding domain families CBM25 and CBM26

Starch binding domains (SBDs) are able to bind to and facilitate the degradation of raw starch and starchy substrates. In general, in the CAZy database they have been classified among the carbohydrate-binding module (CBM) families. The two families CBM25 and CBM26 together with families CBM20, 21, 34, 41, 45, 48, 53, 58, 68 and 69 belong to twelve SBD CAZy families. They represent a group of closely related modules exhibiting some sequence similarity, although each of the two families possesses its own features. Both CBM25 and CBM26 adopt a typical SBD fold of distorted β-barrel as recognized in the modules present in the maltohexaose-producing amylase from Bacillus halodurans. With regard to catalytic domains, most members are α-amylases and maltooligosaccharide-producing amylases from the α-amylase glycoside hydrolase (GH) family GH13, but also some β-amylases (GH14) and hypothetical proteins (e.g. from the family GH31) are known. The main goal of this review was to compare the available amino acid sequences of SBDs from both families CBM25 and CBM26 and to reveal, if possible, SBD(s) with the character “intermediary” between the CBM25 and CBM26. Emphasis was also given on a structural comparison of the identified intermediary SBD with the CBM25 and CBM26 representatives and a detailed evolutionary division of both CBM families that can be utilized for defining the future subfamilies.

Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Using structure to inform carbohydrate binding module function.

Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to the recent emergence of several new CBM families that display diverse binding profiles and novel functions, elucidating the function of these protein modules has become a much more challenging task. This review summarizes several approaches that have been reported for using primary structure to inform CBM specificity and streamlining their biophysical characterization. In addition we discuss general trends in binding site architecture and several newly identified functions for CBMs. Streams of investigation that will facilitate the development and refinement of sequence-based prediction tools are suggested.

Characterization and optimization of heterologous expression in Escherichia coli of the chitinase encoded by the chiA gene of Bacillus halodurans C-125

The diversity of the biotechnological applications of chitinolytic enzymes requires different enzyme-producing strains with different properties suitable for each process. In this work the chitinase encoded by the chiA gene of Bacillus halodurans has been studied. The protein shows a modular structure characterized by the catalytic domain of glycosyl hydrolases family 18 (GH18), fibronectin type III domain (FnIIID) and a carbohydrate-binding module family 5 (CBM5). The expression of the gene in Escherichia coli has made it possible to demonstrate the functionality of the protein which is active in the temperature range of 5–55°C and pH values of 5.5–8.5 while maintaining a high stability under suboptimal conditions. The enzyme hydrolyzes colloidal chitin and different p-NP(GlcNAc)n (n=1–3) by an “-exo” type mechanism according to the information deduced from its sequence. The production of the protein was optimized by constructing recombinant strains, and the effect of the expression vector used, the cell density of the culture, the concentration of inducer and the induction time were studied. Based on its spectrum of activity, stability and mechanism of action, it arises as an enzyme of potential interest for production of N-acetyglucosamine or conversion of chitin into biologically active chito-oligosaccharides.

A new acidophilic endo-β-1,4-xylanase from Penicillium oxalicum: cloning, purification, and insights into the influence of metal ions on xylanase activity.

A new acidophilic xylanase (XYN11A) from Penicillium oxalicum GZ-2 has been purified, identified and characterized. Synchronized fluorescence spectroscopy was used for the first time to evaluate the influence of metal ions on xylanase activity. The purified enzyme was identified by MALDI TOF/TOF mass spectrometry, and its gene (xyn11A) was identified as an open reading frame of 706bp with a 68bp intron. This gene encodes a mature protein of 196 residues with a predicted molecular weight of 21.3kDa that has the 100% identity with the putative xylanase from the P. oxalicum 114-2. The enzyme shows a structure comprising a catalytic module family 10 (GH10) and no carbohydrate-binding module family. The specific activities were 150.2, 60.2, and 72.6 U/mg for beechwood xylan, birchwood xylan, and oat spelt xylan, respectively. XYN11A exhibited optimal activity at pH 4.0 and remarkable pH stability under extremely acidic condition (pH 3). The specific activity, K m and V max values were 150.2 U/mg, 30.7mg/mL, and 403.9μmol/min/mg for beechwood xylan, respectively. XYN11A is a endo-β-1,4-xylanase since it release xylobiose and xylotriose as the main products by hydrolyzing xylans. The activity of XYN11A was enhanced 155% by 1mM Fe(2+) ions, but was inhibited strongly by Fe(3+). The reason of enhancing the xylanase activity of XYN11A with 1mM Fe(2+) treatment may be responsible for the change of microenvironment of tryptophan residues studied by synchronous fluorescence spectrophotometry. Inhibition of the xylanase activity by Fe(3+) was first time demonstrated to associate tryptophan fluorescence quenching.

Overproduction, purification, crystallization and preliminary X-ray characterization of the family 46 carbohydrate-binding module (CBM46) of endo-β-1,4-glucanase B (CelB) from Bacillus halodurans.

Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-terminal family 46 CBM module (BhCBM46) of Bacillus halodurans endo-β-1,4-glucanase B (CelB) was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. Preliminary structural characterization was carried out on BhCBM46 crystallized in different conditions. The crystals of BhCBM46 belonged to the tetragonal space group I4₁22. Data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively. The BhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite.

Biochemical properties and atomic resolution structure of a proteolytically processed β-mannanase from cellulolytic Streptomyces sp. SirexAA-E.

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)8-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite -8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

A starch‐binding domain identified in α‐amylase (AmyP) represents a new family of carbohydrate‐binding modules that contribute to enzymatic hydrolysis of soluble starch

A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyPΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyPΔSBD.

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-L-arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-L-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp(202) and Glu(361) were catalytic residues, and Trp(270), Tyr(461), and Asn(462) were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn(462) and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.

A Noncellulosomal Mannanase26E Contains a CBM59 inClostridium cellulovorans

A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence of C. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method. C. cellulovorans was cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced by C. cellulovorans were separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins from C. cellulovorans were quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium.

Solution structure of the chitin-binding domain 1 (ChBD1) of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase, from Pyrococcus furiosus, is a hyperthermophilic glycosidase that effectively hydrolyses both α and β crystalline chitin. This chitinase has unique structural features; it contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBD1 and ChBD2). We have determined the structure of ChBD1, which significantly enhances the activity of the catalytic domains, by nuclear magnetic resonance spectroscopy. The overall structure of ChBD1 had a compact and globular architecture consisting of three anti-parallel β-strands, similar to those of other proteins classified into carbohydrate-binding module (CBM) family 5. A mutagenesis experiment suggested three solvent-exposed aromatic residues (Tyr112, Trp113 and Tyr123) as the chitin-binding sites. The involvement of Tyr123 or the corresponding aromatic residues in other CBMs, has been demonstrated for the first time. This result indicates that the binding mode may be different from those of other chitin-binding domains in CBM family 5. In addition, the binding affinities of ChBD1 and ChBD2 were quite different, suggesting that the two ChBDs each play a different role in efficiently increasing the activities of AD1 and AD2.

An Unusual Mode of Galactose Recognition by a Family 32 Carbohydrate-Binding Module

Carbohydrate-binding modules (CBMs) are ancillary modules commonly associated with carbohydrate-active enzymes (CAZymes) that function to mediate the adherence of the parent enzyme to its carbohydrate substrates. CBM family 32 (CBM32) is one of the most diverse CBM families, whose members are commonly found in bacterial CAZymes that modify eukaryotic glycans. One such example is the putative μ-toxin, CpGH84A, of the family 84 glycoside hydrolases, which comprises an N-terminal putative β-N-acetylglucosaminidase catalytic module and four tandem CBM32s. Here, we report a unique mode of galactose recognition by the first CBM32, CBM32-1 from CpGH84A. Solution NMR-based analyses of CpGH84A CBM32-1 indicate a divergent subset of residues, located in ordered loops at the apex of the CBM, conferring specificity for the galacto-configured sugars galactose, GalNAc, and LacNAc that differs from those of the canonical galactose-binding CBM32s. This study showcases the impressive variability in ligand binding by this CBM family and offers insight into the growing role of these modules in the interaction of CAZymes with eukaryotic glycans.

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white- and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding cellulases belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and cellulases in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulase and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 β-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.