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PubMed2006

Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins.

Family GH13, also known as the alpha-amylase family, is the largest sequence-based family of glycoside hydrolases and groups together a number of different enzyme activities and substrate specificities acting on alpha-glycosidic bonds. This polyspecificity results in the fact that the simple membership of this family cannot be used for the prediction of gene function based on sequence alone. In order to establish robust groups that show an improved correlation between sequence and enzymatic specificity, we have performed a large-scale analysis of 1691 family GH13 sequences by combining clustering, similarity search and phylogenetic methods. About 80% of the sequences could be reliably classified into 35 subfamilies. Most subfamilies appear monofunctional (i.e. contain enzymes with the same substrate and the same product). The close examination of the other, apparently polyspecific, subfamilies revealed that they actually group together enzymes with strongly related (or even sometimes virtually identical) activities. Overall our subfamily assignment allows to set the limits for genomic function prediction on this large family of biologically and industrially important enzymes.

Amino Acid Sequence↗

PubMed2006

Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4.

"Classical" glycosidases utilize either direct or double-displacement mechanisms involving oxocarbenium ion-like transition states to catalyze the hydrolysis of glycosidic bonds. By contrast, the mechanism of the glycosidases in glycoside hydrolase family 4 has been recently proposed to involve NAD+-mediated redox steps along with alpha,beta-elimination and addition steps via anionic intermediates. Support for this mechanism in BglT, a 6-phospho-beta-glucosidase in family 4, has been provided through mechanistic and X-ray crystallographic analyses [Yip, V. L.Y., et al. (2004) J. Am. Chem. Soc. 126, 8354-8355] in which primary deuterium kinetic isotope effects for the hydride abstraction at C3 and for the alpha-proton abstraction at C2 indicate that these two steps are both partially rate-limiting. Current data reveal that there is no secondary deuterium kinetic isotope effect associated with the rehybridization of the C1 sp3 center to a sp2 center. Furthermore, a flat linear free energy relationship was established with a series of aryl 6-phospho-beta-D-glucosides of varying leaving group abilities. Taken together, these data indicate that cleavage of the C1-O1 linkage does not occur during a rate-limiting step. Since the deprotonation at C2 is slow and partially rate-limiting while the departure of the leaving group is not, a stepwise E1(cb)-type mechanism rather than an E1 or a concerted E2-syn mechanism is proposed. Direct evidence for the role of NAD+ was obtained by reduction in situ using NaBH4 leading to an inactive enzyme that could be reactivated by the addition of excess NAD+. This was accompanied by the expected UV-vis spectrophotometric changes.

Catalysis↗

PubMed2000

A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases.

The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G- x(5,11)-D. This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80. It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp. N174. In particular, it includes two carboxylic residues directly involved in catalysis. We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family.

Amino Acid Sequence↗

PubMed2000

Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10.

A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives. The effects of the aglycone chainlength can clearly be described. Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation. MS measurements showed 1:1 stoichiometries inmost labelling experiments. Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions.

Alkylation↗

PubMed2006

Optimum substrate size and specific anomer requirements for the reducing-end glycoside hydrolase di-N-acetylchitobiase.

Di-N-acetylchitobiase is a family 18 glycoside hydrolase that splits the reducing-end GlcNAc from chitooligosaccharides. The enzyme hydrolyzed only the alpha-anomer of five tested substrates, chitin di- through hexasaccharide. In all cases the glycosyl fragment retained its beta-configuration while the split monosaccharide was alpha-D-GlcNAc. Chitobiose was hydrolyzed less than half as fast as the other larger substrates. All four of them, tri- to hexasaccharide, reacted at the same rate. The biochemical behavior of di-N-acetylchitobiase indicates it has three subsites, -2, -1, +1, in which the reducing-end trimer of any sized chitooligosaccharide is bound. The +1 site is specific for an alpha-anomer.

Acetylglucosaminidase↗

PubMed2004

Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis.

Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfam database and blast tools. Owing to the sequence heterogeneity of the GH-57 members, sequence alignments were performed using mainly manual methods. Likewise, five conserved regions were identified, which are postulated to be GH-57 consensus motifs. In the 659 amino acid-long 4-alpha-glucanotransferase from Thermococcus litoralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and fourth conserved regions contain the catalytic nucleophile and the proton donor, respectively. Based on our sequence alignment, residues Glu291 and Asp394 were proposed as the nucleophile and proton donor, respectively, in a GH-57 amylopullulanase from Thermococcus hydrothermalis. To validate this prediction, site-directed mutagenesis was performed. The results of this work reveal that both residues are critical for the pullulanolytic and amylolytic activities of the amylopullulanase. Therefore, these data support the prediction and strongly suggest that the bifunctionality of the amylopullulanase is determined by a single catalytic centre. Despite this positive validation, our alignment also reveals that certain GH-57 members do not possess the Glu and Asp corresponding to the predicted GH-57 catalytic residues. However, the sequences concerned by this anomaly encode putative proteins for which no biochemical or enzymatic data are yet available. Finally, the evolutionary trees generated for GH-57 reveal that the entire family can be divided into several subfamilies that may reflect the different enzyme specificities.

Amino Acid Sequence↗

PubMed2006

Docking studies on glycoside hydrolase Family 47 endoplasmic reticulum alpha-(1-->2)-mannosidase I to elucidate the pathway to the substrate transition state.

Alpha-(1-->2)-mannosidase I from the endoplasmic reticulum (ERManI), a Family 47 glycoside hydrolase, is a key enzyme in the N-glycan synthesis pathway. Catalytic-domain crystal structures of yeast and human ERMan1s have been determined, the former with a hydrolytic product and the latter without ligands, with the inhibitors 1-deoxymannojirimycin and kifunensine, and with a thiodisaccharide substrate analog. Both inhibitors were bound at the base of the funnel-shaped active site as the unusual 1C4 conformer, while the substrate analog glycon is a 3S1 conformer. In the current study, AutoDock was used to dock alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose with its glycon in chair (1C4,4C1), half-chair (3H2,3H4,4H3), skew-boat (OS2,3S1,5S1), boat (2,5B,3,OB,B1,4,B2,5), and envelope (3E,4E,E3,E4) conformations into the yeast ERManI active site. Both docked energies and forces on docked ligand atoms were calculated to determine how the ligand distorts to the transition state. From these, we can conclude that (1) both 1C4 and OS2 can be the starting conformers; (2) the most likely binding pathway is 1C4-->3H2-->OS2-->3,OB-->3S1-->3E; (3) the transition state is likely to be close to a 3E conformation.

Binding Sites↗

PubMed2006

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of Acetivibrio cellulolyticus and Clostridium thermocellum.

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.

Amino Acid Sequence↗

PubMed2004

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.

Biopolymers↗

PubMed2003

Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6.

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.

Amino Acid Substitution↗

PubMed2002

Beta-helical catalytic domains in glycoside hydrolase families 49, 55 and 87: domain architecture, modelling and assignment of catalytic residues.

X-ray crystallography and bioinformatics studies reveal a tendency for the right-handed beta-helix domain architecture to be associated with carbohydrate binding proteins. Here we demonstrate the presence of catalytic beta-helix domains in glycoside hydrolase (GH) families 49, 55 and 87 and provide evidence for their sharing a common evolutionary ancestor with two structurally characterized GH families, numbers 28 and 82. This domain assignment helps assign catalytic residues to each family. Further analysis of domain architecture reveals the association of carbohydrate binding modules with catalytic GH beta-helices, as well as an unexpected pair of beta-helix domains in GH family 55.

Amino Acid Sequence↗

PubMed2001

Mapping of genes encoding glycoside hydrolases on the chromosome of Cellulomonas fimi.

Cellulomonas fimi genomic DNA was digested with HpaI, MunI, HindIII, and NsiI, producing fragments ranging in size from 20 to 1400 kbp that were resolved by pulsed field gel electrophoresis. Genetic and physical linkages were determined by Southern blotting and were used to construct a genome map. Cellulomonas fimi has a single circular chromosome of approx. 4000 kbp. Except for two closely linked genes, cbh6A and cel5A, the genes known to encode glycoside hydrolases are scattered widely on the chromosome.

Actinomycetales↗

PubMed2001

The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity.

To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P. cellulosa genomic DNA was screened for 4-methylumbelliferyl-alpha-L-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000 Da. The substrate specificity of the Abf51A showed that it preferentially removed alpha1,2- and alpha1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed alpha1,5-linked arabino-oligosaccharides, although at a much lower rate. The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P. cellulosa gene. Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively. To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated. The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A. These data, in conjunction with the results presented in the accompanying paper [Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem. J. 358, 599-605], indicate that P. cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.

Amino Acid Sequence↗

PubMed2002

Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A.

The three-dimensional structure of Aspergillus aculeatus beta-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (beta/alpha)(8) barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate-enzyme interactions were elucidated by modeling of beta-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A comparison with the substrate binding grooves of other Clan GH-A enzymes suggests that shape complementarity is crucial in determining the specificity of polysaccharidases.

Aspergillus↗

PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5.

Crystals of the catalytic core domain of a Trichoderma reesei beta-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 A, beta = 111.3 degrees, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 A resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 A at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.

Catalysis↗

PubMed1987

Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates.

The rumen fungi Neocallimastix patriciarum, Piromonas communis, and a morphologically distinct but unidentified isolate were cultivated on the polysaccharides starch, cellulose, xylan, and their principal component monosaccharides and disaccharides, and the range and specific activities of the glycoside hydrolases formed were monitored using gluco-oligosaccharide and p-nitrophenyl glycoside substrates. A wide range of enzyme activities was detected in preparations from vegetative growth and zoospores of all three isolates. Enzyme activity was also present in the culture medium. The specific activities were affected by the carbohydrate source available in the growth medium, although the more active hydrolases involved in the degradation of plant structural and storage polysaccharides were formed on all seven carbohydrate sources evaluated. Enzyme activities were increased in the zoospore, vegetative, and extracellular preparations after growth on the appropriate structurally related disaccharide or polysaccharide. The hemicellulolytic glycosidases (alpha-L-arabinofuranosidase, beta-D-xylosidase) were most active after growth on xylan, whereas alpha-/beta-glucosidase activity was increased with the corresponding glucan as growth substrate. However, whereas wide-ranging beta-glucosidase activity was detected following growth on maltose or starch, the alpha-glucosidase activities of P. communis were lower or undetectable in vegetative preparations grown on glucose or the beta-glucans cellobiose and cellulose.

Animals↗

PubMed2005

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases.

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.

Calmodulin↗

PubMed2005

Hydrolysis of beta-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-beta-glucanase from the basidiomycete Phanerochaete chrysosporium.

When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for beta-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1->6)-beta-D-Glcp-(1->3)-beta-D-Glcp-(1->3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1->4)-beta-D-Glcp-(1->3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes beta-D-Glcp-(1->3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.

Amino Acid Sequence↗