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At least 19 recordsLinked to original sources
PubMed1977

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Animals↗
PubMed1978

Acid hydrolases in blister fluid. II. Characterization and quantification of glycoside hydrolases.

Seven glycoside hydrolases have been investigated in suction blister fluid, interstitial fluid and in serum. Six of these have been characterized; no differences could be demonstrated between the corresponding enzymes from the various sources. The remaining enzyme (beta-glucosidase) was not found. Quantitative data suggest that 2 enzymes (beta-acetylglucosaminidase and beta-glucuronidase) diffuse freely from the epidermis into blister fluid, whereas 4 (alpha-glucosidase, alpha- and beta-galactosidase and alpha-mannosidase) are almost entirely retained in the roof of the bulla.

Blister↗
PubMed2000

The thermostable alpha-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial alpha-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase.

An alpha-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60 degrees C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p-nitrophenyl-alpha-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg-1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1H-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable beta-glucosidase. The secondary structure of RamA consists of 27% alpha-helices and 50% beta-sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases.

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.

Amino Acid Motifs↗
PubMed1980

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.

Acetylglucosaminidase↗
PubMed2001

Mixed-linkage cellooligosaccharides: a new class of glycoside hydrolase inhibitors.

A new class of inhibitors for beta-D-glycoside hydrolases, in which a single alpha-(1-->4)-glycosidic bond is incorporated into an otherwise all-beta-(1-->4)-linked oligosaccharide, is described. Such mixed beta/alpha-linkage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning either side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed alpha/beta-D-tetrasaccharide acts competitively on a number of cellulases, displaying inhibition constants in the range of 40-300 microM. Using the Bacillus agaradhaerens enzyme Cel5A as a model system, one such mixed beta/alpha-cellooligosaccharide, methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside, displays a K(i) value of 100 microM, an inhibition at least 150 times better than is observed with an equivalent all-beta-linked compound. The three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside has been determined at 1.8 A resolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the (4)C(1) chair conformation, occupies the -3, -2 and +1 subsites whilst evading the catalytic (-1) subsite. Such "by-pass" compounds offer great scope for the development of a new class of beta-D-glycoside hydrolase inhibitors.

Bacillus↗
PubMed2001

Novel, thermostable family-13-like glycoside hydrolase from Methanococcus jannaschii.

A novel glycoside hydrolase from the hyperthermophilic archaeon Methanococcus jannaschii has been cloned into Escherichia coli. Extremely thermoactive and thermostable amylolytic activity was confirmed in partially purified enzyme solution. This enzyme exhibited a temperature optimum of 100 degrees C and a pH optimum pH 5.0-8.0. Hydrolysis of large 1,6-alpha- and 1,4-alpha-linked polysaccharides yielded glucose polymers of 1-7 units. Incubation with amylose displayed the highest activity. The catalyst was activated and stabilized by Ca2+ and exhibited extreme thermostability at 100 degrees C with a half-life of 78 h.

Amino Acid Sequence↗
PubMed2003

Radiation and functional specialization of the family-3 glycoside hydrolases.

A phylogenetic analysis of the glycoside hydrolases of family 3 (GH3s) was conducted in order to infer particular trends in its evolution: functional specialization, gene transfer events, gene duplications and paralogous evolution, and gene deletions. The phylogenetic analysis of GH3s revealed six clusters, i.e., A, B, C, D, E, and F that could fit the definition of 3 sub-families, i.e., AB, AB' and AB". While the sub-families AB' and AB" contain a single cluster, F and E, respectively, the AB sub-family is sub-divided into four clusters. Global analysis of the GH3 phylogenetic tree suggests a primary burst of amplification of the GH3s that might have led to these sub-families. Specializations, gene transfers, and gene duplications among each of these sub-families and phylogenetic clusters might then have occurred and have been inferred. The fine comparison of the enzyme properties and phylogenetic relationships of GH3s allowed to detect common functional groups that belong to the same cluster (D, E or F), or sub-cluster (A1, A2 or B2). The prokaryotic and eukaryotic beta-xylosidases and beta-glucosidases belong to the AB and AB' sub-families, and the N-acetylglucosaminidases are in sub-family AB" (in cluster E). In some instances (B1, B2, C1, C2, and C3), the lack of data and/or the high heterogeneity of the hydrolytic properties did not allow to infer a particular link between an enzyme functional group and a phylogenetic cluster, suggesting the emergence of some highly specialized GH3s.

Computational Biology↗
PubMed1980

Treatment of non-insulin dependent diabetic adults with a new glycoside hydrolase inhibitor (Bay g 5421).

The hypoglycemic effect of a glycoside hydrolase inhibitor (BAY g 5421) was tested in 10 non-insulin dependent, overweight diabetics by means of continuous in vivo glucograms. There was a highly significant reduction of serum glucose with a dosage of 3 x 100 mg glycoside hydrolase inhibitor, after dosage division to 6 x 50 mg there was an additional reduction of blood glucose. This difference, however, could not be determined statistically. Insulin, glucagon and lactate as well as the triglyceride concentration remained unchanged. There were no significant side-effects. Two of the 10 patients complained of meteorism, after specific questioning three more patients offered similar complaints. Discontinuation of the preparation was not necessary.

Acarbose↗
PubMed1998

The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases.

We report here cloning from the marine gliding bacterium Cytophaga drobachiensis of kappa-carrageenase, a glycoside hydrolase involved in the degradation of kappa-carrageenan. Structural features in the nucleotide sequence are pointed out, including the presence of an octameric omega sequence similar to the ribosome-binding sites of various eukaryotes and prokaryotes. The cgkA gene codes for a protein of 545 aa, with a signal peptide of 35 aa and a 229-aa-long posttranslationaly processed C-terminal domain. The enzyme displays the overall folding and catalytic domain characteristics of family 16 of glycoside hydrolases, which comprises other beta-1,4-alpha-1,3-D/L-galactan hydrolases, beta-1,3-D-glucan hydrolases (laminarinases), beta-1,4-1,3-D-glucan hydrolases (lichenases), and beta-1,4-D-xyloglucan endotransglycosylases. In order to address the origin and evolution of CgkA, a comprehensive phylogenetic tree of family 16 was built using parsimony analysis. Family-16 glycoside hydrolases cluster according to their substrate specificity, regardless of their phylogenetic distribution over eubacteria and eukaryotes. Such a topology suggests that the general homology between laminarinases, agarases, kappa-carrageenases, lichenases, and xyloglucan endotransglycosylases has arisen through gene duplication, likely from an ancestral protein with laminarinase activity.

Amino Acid Sequence↗
PubMed2000

Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases.

There are approximately 100 known members of the family 3 group of glycoside hydrolases, most of which are classified as beta-glucosidases and originate from microorganisms. The only family 3 glycoside hydrolase for which a three-dimensional structure is available is a beta-glucan exohydrolase from barley. The structural coordinates of the barley enzyme is used here to model representatives from distinct phylogenetic clusters within the family. The majority of family 3 hydrolases have an NH(2)-terminal (alpha/beta)(8) barrel connected by a short linker to a second domain, which adopts an (alpha/beta)(6) sandwich fold. In two bacterial beta-glucosidases, the order of the domains is reversed. The catalytic nucleophile, equivalent to D285 of the barley beta-glucan exohydrolase, is absolutely conserved across the family. It is located on domain 1, in a shallow site pocket near the interface of the domains. The likely catalytic acid in the barley enzyme, E491, is on domain 2. Although similarly positioned acidic residues are present in closely related members of the family, the equivalent amino acid in more distantly related members is either too far from the active site or absent. In the latter cases, the role of catalytic acid is probably assumed by other acidic amino acids from domain 1.

Amino Acid Sequence↗
PubMed2004

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase.

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.

Catalysis↗
PubMed2026

Unlocking the molecular engineering of Geobacillus glycoside hydrolases as a source of industrial biocatalysts.

This review examines Geobacillus sensu stricto as a source of thermostable glycoside hydrolases (GH) for biomass conversion, food processing, and enzyme engineering. Recent peer-reviewed literature was assessed with emphasis on taxonomy, genome-based Carbohydrate-Active Enzymes (CAZyme) prediction, biochemical validation, structural data, and engineering case studies. Taxonomic boundaries were interpreted using current Anoxybacillaceae frameworks, with Parageobacillus treated as a related comparator rather than as Geobacillus. The strongest evidence supports GH13 alpha-amylases, xylan-active systems, beta-xylosidases, and selected accessory enzymes. Recent studies also show that genome mining must be coupled with enzymatic assays and product profiling because CAZyme annotation alone does not prove industrial function. Molecular engineering has improved relevant traits, including the longer thermal half-life of engineered G. stearothermophilus alpha-amylase variants, the increased catalytic efficiency of oligo-alpha-1,6-glucosidase variants, and improved AmyS expression in Bacillus subtilis. Geobacillus glycoside hydrolases are best interpreted as process-specific, engineerable biocatalytic templates. Their translation requires reliable taxonomy, functional validation, structural interpretation, scalable expression and testing on realistic substrates. This synthesis also recognises current limitations: many predicted CAZymes still lack biochemical validation, complete cellulolytic systems remain less mature than xylan- and starch-active systems, and scale-up data remain scarce.

Geobacillus↗
PubMed2003

First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis.

The room-temperature structure of xylanase (EC 3.2.1.8) from the bacterial plant pathogen Erwinia chrysanthemi expressed in Escherichia coli, a 45 kDa, 413-amino acid protein belonging to glycoside hydrolase family 5, has been determined by multiple isomorphous replacement and refined to a resolution of 1.42 A. This represents the first structure of a xylanase not belonging to either glycoside hydrolase family 10 or family 11. The enzyme is composed of two domains similar to most family 10 xylanases and the alpha-amylases. The catalytic domain (residues 46-315) has a (beta/alpha)(8)-barrel motif with a binding cleft along the C-terminal side of the beta-barrel. The catalytic residues, Glu165 and Glu253, determined by correspondence to other family 5 and family 10 glycoside hydrolases, lie inside this cleft on the C-terminal ends of beta-strands 4 and 7, respectively, with an O(epsilon)2...O(epsilon)1 distance of 4.22 A. The smaller domain (residues 31-43 and 323-413) has a beta(9)-barrel motif with five of the strands interfacing with alpha-helices 7 and 8 of the catalytic domain. The first 13 N-terminal residues form one beta-strand of this domain. Residues 44, 45, and 316-322 form the linkers between this domain and the catalytic domain.

Amino Acid Sequence↗
PubMed1997

Structural and sequence-based classification of glycoside hydrolases.

The diversity of oligo- and polysaccharides provides an abundance of biological roles for these carbohydrates. The enzymes hydrolysing these compounds, the glycoside hydrolases, therefore mediate a wealth of biological functions. Glycoside hydrolases fall into a number of sequence-based families. The recent analysis of these families, coupled with the burgeoning number of 3D structures, provides a detailed insight into the structure, function and catalytic mechanism of these enzymes.

Amino Acid Sequence↗
PubMedDate not supplied

Glycoside hydrolase production by an anaerobic rumen fungus Caecomyces communis.

The ruminal fungus Caecomyces communis was grown anaerobically either in a discontinuous cultivation system or in a fermentor with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The best culture conditions for glycoside hydrolase production were obtained in Lowe medium with daily fresh medium addition, whereas the Orpin medium with ruminal fluid was favourable to fungal growth and to the enzyme export process. Among glycoside hydrolases assessed in both culture fluid and cellular homogenate, beta-D-fucosidase activity was preponderant. Most studied enzymes were mainly associated with cells (from 50% to 99%). Glycoside hydrolase activities were constitutive, but their level was regulated by a carbon source. beta-D-fucosidase and beta-D-xylosidase activity production was activated by the association of glucose plus cellobiose, whereas beta-D-glucosidase activity production was stimulated by cellobiose alone. Enzyme release could be favoured by glucose alone or by Ray grass hay added to glucose plus cellobiose.

Anaerobiosis↗
PubMed2000

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

Glycoside Hydrolases↗
PubMed2001

Glycoside hydrolases and glycosyltransferases: families and functional modules.

The past year has witnessed the expected increase in the number of solved structures of glycoside hydrolases and glycosyltransferases, and their constitutive modules. These structures show that, while glycoside hydrolases display an extraordinary variety of folds, glycosyltransferases and carbohydrate-binding modules appear to belong to a much smaller number of folding families.

Animals↗