The modification of N-glycans by ␣-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for ␣1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-␣1,6-mannosidase activity consistent with specificity for N-linked glycans having their ␣1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substratebound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (␣/␣) 6 -fold, recognize and hydrolyze the ␣1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-␣1,6-mannosidases.A feature of emerging importance to bacteria that colonize or infect humans is their capacity to process host glycans. Streptococcus pneumoniae is one notable human pathogen that relies on this ability for its full virulence (1). Among its known carbohydrate active virulence factors are NanA, StrH, BgaA, and EndoD. NanA Glycoside hydrolases, enzymes that break glycosidic bonds through a hydrolytic mechanism, are presently classified into 123-amino acid sequence based families (2). ␣-Mannosidases known to process N-glycans are found in families 38, 47, 76, 92, and 99. Very recent studies have shown the bacterial family 38 ␣-mannosidase from Streptococcus pyogenes (SpyGH38) to be a specific exo-␣1,3-mannosidase that is tolerant of the ␣1,6-branches in N-glycans (3). Analysis of family 92 glycoside hydrolases from the human gut symbiont Bacteroides thetaiotaomicron revealed an expanded repertoire of ␣-mannosidases (4). These enzymes displayed activity primarily toward ␣1,2-and ␣1,3-mannosidic linkages with some having low ␣1,6-mannosidase activity. In addition to the established ability of S. pneumoniae to exo-hydrolytically process the distal arms of complex glycans, which comprise sialic acid, galactose, and N-acetylglucosamine, consideration of additional putative carbohydrate-active enzymes found in this organism suggests it can partly degrade the mannose component of N-glycans using enzymes similar to those found in S. pyogenes and B. thetaiotaomicron. Through these observations it has become clear that some bacteria, possibly including S. pneumoniae, have the capacity to process the mannose component of N-glycans. A noteworthy gap, however, in the known bacterial N-glycan de...

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 ␣-fucosidase and from a family 98 blood group A/B antigen-specific endo-␤-galactosidase, respectively, showed them to have highly similar ␤-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.Carbohydrates have critical functions in numerous biological events, including, for example, the movement and interactions of cells and proteins in animals, the recycling of plant cell wall carbohydrates, and the interactions between hosts and disease-causing organisms. Central to the role of carbohydrates in biological processes are protein-carbohydrate interactions. Non-catalytic carbohydrate-binding proteins (e.g. lectins, antibodies, and transport proteins) and catalytic carbohydrate-active enzymes are finely tuned to recognize particular carbohydrate structural motifs. The information content of glycans is realized through the specificity of non-catalytic carbohydratebinding proteins (like lectins and antibodies), whereas carbohydrate-active enzymes change the information content and often unlock the energy contained within these molecules.Carbohydrate-binding modules (CBMs) 3 are a comparatively new class of non-catalytic carbohydrate-recognizing polypeptide that are generally defined by their presence as ancillary modules in larger, multimodular carbohydrate-active enzymes such as glycoside hydrolases, glycosyltransferases, and polysaccharide lyases (1). In the context of these enzymes, the role of CBMs is to specifically bind the carbohydrate substrate and hold the enzyme in proximity to the substrate, allowing catalysis to proceed more efficiently (2). The number of CBM families, which are defined on the basis of amino acid sequence similarity, has grown to the current number of 50 (www.cazy. org). ...

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the -toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (K a ‫؍‬ 1.44 ؋ 10 11 M ؊1 ) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrateactive enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The -toxin dockerin module in this complex is positioned Ϸ180°relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.enzyme complexes ͉ glycoside hydrolases ͉ protein-protein interaction

Background:The endo-␣-D-N-acetylgalactosaminidase SpGH101 from Streptococcus pneumoniae hydrolyzes the O-linked T-antigen from proteins. Results: SpGH101 displays an unusual conformational change on substrate binding and a distinctive arrangement of its catalytic machinery. Conclusion: Substrate hydrolysis proceeds through a retaining mechanism with a proton shuttle. Significance: This is the first evidence of proton shuttle in a retaining glycoside hydrolase.

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens -toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.