Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

Identification and characterization of a novel bacterial β-glucosidase that is highly specific for the β-1,2-glucosidic linkage of sesaminol triglucoside

A gene (PSTG2) coding for a novel β-glucosidase belonging to glycoside hydrolase family 3 was identified in the vicinity of the previously identified β-glucosidase gene [sesaminol triglucoside (STG)-hydrolyzing β-glucosidase, PSTG1] in the genome of Paenibacillus sp. strain KB0549. Compared with PSTG1, recombinant PSTG2 more specifically acted on the β-1,2-glucosidic linkage of the STG molecule to transiently accumulate a larger amount of 6-O-(β-D-glucopyranosyl)-β-D-glucopyranosylsesaminol.     

Characterization of a novel β-L-Arabinofuranosidase in Bifidobacterium longum: functional elucidation of A DUF1680 family member

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.     

The structural basis of substrate recognition in an exo-beta-D-glucosaminidase involved in chitosan hydrolysis

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with β-galactosidase activity (Escherichia coli LacZ), β-glucuronidase activity (Homo sapiens GusB), and β-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-β-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 Å resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural β-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-β-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.     

Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate

Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, 4-nitrophenyl β-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-β-D-glucosaminide, peptidoglycan or 4-nitrophenyl β-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the β-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galβ1 → 4GlcNAcβ-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose

We solved the 1.8 ? crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.     

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.     

A New Archaeal β-Glycosidase from Sulfolobus solfataricus: SEEDING A NOVEL RETAINING β-GLYCAN-SPECIFIC GLYCOSIDE HYDROLASE FAMILY ALONG WITH THE HUMAN NON-LYSOSOMAL GLUCOSYLCERAMIDASE GBA2*

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl β-gluco- and β-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid β-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes β-glucosidases (EC 3.2.1.21), β-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.     

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum: FUNCTIONAL ELUCIDATION OF A DUF1680 PROTEIN FAMILY MEMBER*

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. Previously, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-338 is a critical residue for catalytic activity. This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127.     

Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.     

Biochemical analysis of cross-feeding behaviour between two common gut commensals when cultivated on plant-derived arabinogalactan

In this paper, we reveal and characterize cross-feeding behaviour between the common gut commensal Bacteroides cellulosilyticus (Baccell) and certain bifidobacterial strains, including Bifidobacterium breve UCC2003, when grown on a medium containing Larch Wood Arabinogalactan (LW-AG). We furthermore show that cross-feeding is dependent on the release of β-1,3-galacto-di/trisaccharides (β-1,3-GOS), and identified that the bga gene cluster of B. breve UCC2003 allows β-1,3-GOS metabolism. The product of bgaB is presumed to be responsible for the import of β-1,3-GOS, while the bgaA gene product, a glycoside hydrolase family 2 member, was shown to hydrolyse both β-1,3-galactobiose and β-1,3-galactotriose into galactose monomers. This study advances our understanding of strain-specific syntrophic interactions between two glycan degraders in the human gut in the presence of AG-type dietary polysaccharides.     

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.     

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase

Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.     

Association of novel domain in active site of archaic hyperthermophilic maltogenic amylase from Staphylothermus marinus

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.     

Structure and activity of exo-1,3/1,4-β-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain

Following the discovery of an exo-1,3/1,4-β-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 ? resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue β-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX.     

Crystal structure of hyperthermophilic endo-β-1,4-glucanase: implications for catalytic mechanism and thermostability

Endo-β-1,4-glucanase from thermophilic Fervidobacterium nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase family 5, is highly thermostable and exhibits the highest activity on carboxymethylcellulose among the reported homologues. To understand the structural basis for the thermostability and catalytic mechanism, we report here the crystal structures of FnCel5A and the complex with glucose at atomic resolution. FnCel5A exhibited a (β/α)(8)-barrel structure typical of clan GH-A of the glycoside hydrolase families with a large and deep catalytic pocket located in the C-terminal end of the β-strands that may permit substrate access. A comparison of the structure of FnCel5A with related structures from thermopile Clostridium thermocellum, mesophile Clostridium cellulolyticum, and psychrophile Pseudoalteromonas haloplanktis showed significant differences in intramolecular interactions (salt bridges and hydrogen bonds) that may account for the difference in their thermostabilities. The substrate complex structure in combination with a mutagenesis analysis of the catalytic residues implicates a distinctive catalytic module Glu(167)-His(226)-Glu(283), which suggests that the histidine may function as an intermediate for the electron transfer network between the typical Glu-Glu catalytic module. Further investigation suggested that the aromatic residues Trp(61), Trp(204), Phe(231), and Trp(240) as well as polar residues Asn(51), His(127), Tyr(228), and His(235) in the active site not only participated in substrate binding but also provided a unique microenvironment suitable for catalysis. These results provide substantial insight into the unique characteristics of FnCel5A for catalysis and adaptation to extreme temperature.     

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.