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Hydrolase Family (hydrolase + family)
Kinds of Hydrolase Family Selected AbstractsEmergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18FEBS JOURNAL, Issue 7 2005Anne Durand The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibitors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and predicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 ,inactivated' chitinases. [source] Xylanases, xylanase families and extremophilic xylanasesFEMS MICROBIOLOGY REVIEWS, Issue 1 2005Tony Collins Abstract Xylanases are hydrolytic enzymes which randomly cleave the , 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. [source] Analysis of functional divergence within two structurally related glycoside hydrolase familiesBIOPOLYMERS, Issue 6 2009Blake Mertz Abstract Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared ,-jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3-,- D -glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 478,495, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Glycoside hydrolases: Catalytic base/nucleophile diversityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Thu V. Vuong Abstract Recent studies have shown that a number of glycoside hydrolase families do not follow the classical catalytic mechanisms, as they lack a typical catalytic base/nucleophile. A variety of mechanisms are used to replace this function, including substrate-assisted catalysis, a network of several residues, and the use of non-carboxylate residues or exogenous nucleophiles. Removal of the catalytic base/nucleophile by mutation can have a profound impact on substrate specificity, producing enzymes with completely new functions. Biotechnol. Bioeng. 2010;107: 195,205. © 2010 Wiley Periodicals, Inc. [source] Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detectionBIOTECHNOLOGY JOURNAL, Issue 5 2007Mikhail L. Rabinovich Professor Abstract Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine. [source] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltoseFEBS JOURNAL, Issue 20 2010Keizo Yamamoto The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (,/,)8 -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310,315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively. [source] Aspergillus nidulans,-galactosidase of glycoside hydrolase family 36 catalyses the formation of ,-galacto-oligosaccharides by transglycosylationFEBS JOURNAL, Issue 17 2010Hiroyuki Nakai The ,-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic ,-galactosidases and ,-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L,1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with ,-(1,6) regioselectivity from 40 mm 4-nitrophenol ,- d -galactopyranoside, melibiose or raffinose, resulting in a 37,74% yield of 4-nitrophenol ,- d -Galp -(1,6)- d -Galp, ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp and ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp -(,1,,2)- d -Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol ,- d -galactopyranoside (40 mm), ,-(1,6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39,58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l -arabinose, l -fucose and l -rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l -rhamnose. Structural modelling using Thermotoga maritima GH36 ,-galactosidase as the template and superimposition of melibiose from the complex with human GH27 ,-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred ,-galactosyl to 6-OH of the terminal residue in the ,-linked melibiose, maltose, trehalose, sucrose and turanose in 6,46% yield and the ,-linked lactose, lactulose and cellobiose in 28,38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. ,- d -Galp -(1,6)- d -Manp; ,- d -Galp -(1,6)-,- d -Glcp -(1,4)- d -Glcp; ,- d -Galp -(1,6)-,- d -Galp -(1,4)- d -Fruf; ,- d -Galp -(1,6)- d -Glcp -(,1,,1)- d -Glcp; and ,- d -Galp -(1,6)-,- d -Glcp -(1,3)- d -Fruf. [source] Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymesFEBS JOURNAL, Issue 20 2009Wim Van den Ende Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled. [source] Characterization of chitinase-like proteins (Cg -Clp1 and Cg -Clp2) involved in immune defence of the mollusc Crassostrea gigasFEBS JOURNAL, Issue 14 2007Fabien Badariotti Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg -Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg -Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg -Clp1- and Cg -Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity. [source] Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18FEBS JOURNAL, Issue 7 2005Anne Durand The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibitors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and predicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 ,inactivated' chitinases. [source] Insights into the reaction mechanism of glycosyl hydrolase family 49FEBS JOURNAL, Issue 22 2004Site-directed mutagenesis, substrate preference of isopullulanase Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite ,1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and ,-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities. [source] Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilarisFEBS JOURNAL, Issue 16 2003Masahiro Sugimura A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source] Relation between domain evolution, specificity, and taxonomy of the ,-amylase family members containing a C-terminal starch-binding domainFEBS JOURNAL, Issue 4 2003tefan Jane The ,-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): ,-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic ,-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (,/,)8 -barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the ,complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the ,SBD tree' the border between hydrolases (mainly,-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic ,-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (,/,)8 -barrel(domain A). [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source] Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilizationFEBS JOURNAL, Issue 13 2000Vincent Monchois Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the ,-amylase family (glycoside hydrolase family 13), which all have a (,/,)8 barrel structure. In the glucansucrases, however, the ,-helix and ,-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In ,-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis,Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (,/,)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which ,1,3-linked glucosyl units are joined sequentially to maltose. [source] Functional importance of Asp37 from a family 11 xylanase in the binding to two proteinaceous xylanase inhibitors from wheatFEMS MICROBIOLOGY LETTERS, Issue 1 2004Tariq A. Tahir Abstract Aspergillus niger xylanase is a target enzyme of the two wheat proteinaceous inhibitors, XIP-I and TAXI-I. We previously suggested that the xylanase "thumb" region was XIP-I binding site. Here, we expressed the Asp37Ala mutant in Pichia pastoris and showed that the mutation abolished the enzyme capacity to interact with both inhibitors, suggesting a direct contact at the active site. The mutant pH profile was altered, confirming the key role of Asp37 in determining the pH optima of glycoside hydrolase family 11. The results are consistent with a competitive inhibition mode and underline the strategic importance of Asp37 in the inhibition mechanism. [source] The ,-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombeMOLECULAR MICROBIOLOGY, Issue 5 2008María De Medina-Redondo Summary Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the ,-1,3-glucan synthase bgs2p synthesizes linear ,-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between ,-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with ,-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4+, a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4, diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4+ is strongly induced during sporulation and a yellow fluorescent protein (YFP),gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe. [source] The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitisPROTEIN SCIENCE, Issue 3 2000Thomas Hollis Abstract The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 Å resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded ,/,-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme. [source] Production, purification and characterisation of a novel halostable xylanase from Bacillus sp.ANNALS OF APPLIED BIOLOGY, Issue 2 2010NTU-0 Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0,20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had Km and Vmax values of 3.45 mg mL,1 and 387.3 µmol min,1mg,1, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed. [source] The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site grooveACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Elise Champion Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis,Menten kinetics, with a Km of 60.7,mM and a kcat of 21.7,s,1. The structure of the enzyme was solved at a resolution of 1.9,Å in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite ,1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite ,1 from the `nonreducing' end. Binding of the glucosyl moiety in subsite ,1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the `nonreducing' end during the glucan-elongation cycle. [source] Crystallization and preliminary X-ray analyses of the active and the inactive forms of family GH-8 chitosanase with subclass II specificity from Bacillus sp. strain K17ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Yuri Sakihama Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3,10), but is active in a much narrower pH range (4.5,7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0,Å resolution, respectively. [source] Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex formsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Teruya Nakamura During replication, Escherichia coli MutT prevents the misincorporation of mutagenic 8-oxoguanine into nascent DNA strands opposite adenine by hydrolyzing 8-oxo-dGTP in nucleotide pools to 8-oxo-dGMP. E. coli MutT is the most widely investigated member of the Nudix hydrolase family, which is large and found in all organisms. By co-crystallization of MutT with 8-oxo-dGMP, a reaction product, crystals of the binary complex were obtained using ammonium sulfate as a precipitant. The crystals belong to space group P212121, with unit-cell parameters a = 37.9, b = 56.0, c = 59.4,Å. Assuming the presence of one protein,nucleotide complex in the asymmetric unit, the Matthews coefficient VM is 2.1,Å3,Da,1. Crystals of the ternary complex were prepared by soaking crystals of the binary complex in 1,mM MnCl2 solution. They diffracted to 1.96 and 2.56,Å resolution, respectively. [source] Preparation and preliminary X-ray analysis of the catalytic module of ,-1,3-xylanase from the marine bacterium Vibrio sp.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004,-1,3-xylanase (1,3-,- d -xylan xylanohydrolase; EC 3.2.1.32) is an enzyme capable of hydrolyzing ,-1,3-xylan. The newly cloned ,-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 (XYL4) exhibited a modular structure consisting of three modules: an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two C-terminal xylan-binding modules belonging to carbohydrate-binding module family 31. Despite substantial crystallization screening, crystallization of the recombinant XYL4 was not accomplished. However, the deletion mutant of XYL4, composed of a catalytic module without a xylan-binding module, was crystallized. The crystal belonged to space group P212121, with unit-cell parameters a = 51.6, b = 75.8, c = 82.0,Å. X-ray diffraction data were collected to 1.44,Å resolution. [source] Crystallization and preliminary X-ray analysis of a novel Trichoderma reesei xylanase IV belonging to glycoside hydrolase family 5ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Tarja Parkkinen Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 86.3, b = 137.5, c = 196.1,Å, , = , = , = 90°. Assuming a molecular weight of 50.3,kDa, the VM values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8,Å resolution has been collected at a home laboratory and a data set to 2.2,Å resolution has been collected using synchrotron radiation. [source] The structure of a family GH25 lysozyme from Aspergillus fumigatusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Justyna E. Korczynska Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 `lysozyme' from Aspergillus fumigatus was determined. This is the first fungal structure from the family and reveals a modified ,/,-barrel-like fold in which an eight-stranded ,-barrel is flanked by three ,-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operates via a `substrate-assisted' catalytic mechanism. [source] Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Keisuke Ito Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1,Å resolution. [source] Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Annelies Van Hoorebeke Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [d -glucopyranosyl-,(1,1),- d -glucopyranose] to ,- d -glucose 1-phosphate and d -glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P212121. Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n -octyl-,- d -glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420,Å, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7,8 to 3,4,Å resolution. The structure of recombinant TP was determined by molecular replacement to 2.8,Å resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase. [source] Crystallization and preliminary X-ray crystallographic studies of an exo-,- d -glucosaminidase from Trichoderma reeseiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Yasumitsu Sakamoto Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-,- d -glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93,kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of ,105,kDa (Gls93-F1) and ,100,kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 98.27, b = 98.42, c = 108.28,Å, and diffracted to 1.8,Å resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 81.62, c = 183.14,Å, and diffracted to 2.4,Å resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution. [source] Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 ,-glucosidaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Sompong Sansenya Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 ,-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-,- d -glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1,M Tris,HCl pH 8.5, 0.16,M NaCl at 288,K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45,Å resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P43212 by molecular replacement using the white clover cyanogenic ,-glucosidase structure (PDB code 1cbg) as a search model. [source] Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas udaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Annelies Van Hoorebeke Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [,- d -glucopyranosyl-(1,4)- d -glucopyranose] to ,- d -glucose-1-phosphate and d -glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity. [source] | |||||||||||||||||||||||||