scholarly journals Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases

2006 ◽  
Vol 394 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Nathalie Côté ◽  
Alain Fleury ◽  
Émilie Dumont-Blanchette ◽  
Tamo Fukamizo ◽  
Masaru Mitsutomi ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Cloning And Expression ◽  
Streptomyces Avermitilis ◽  
Gene Cloning And Expression ◽  
Glycoside Hydrolase Family ◽  
Sequence Alignments

A GlcNase (exo-β-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with Km and kcat values of 0.16 mg/ml and 2832 min−1. On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative β-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable β-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with β-galactosidase, β-glucuronidase or β-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.

scholarly journals Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

2018 ◽  
Vol 293 (47) ◽  
pp. 18138-18150 ◽  
Author(s):  
Léa Chuzel ◽  
Mehul B. Ganatra ◽  
Erdmann Rapp ◽  
Bernard Henrissat ◽  
Christopher H. Taron
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Recombinant Expression ◽  
Biochemical Property ◽  
Environmental Dna ◽  
Thermal Spring ◽  
Sialic Acid Residue ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.

scholarly journals Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a β-N-acetylglucosaminidase and not a hyaluronidase

2006 ◽  
Vol 399 (2) ◽  
pp. 241-247 ◽  
Author(s):  
William L. Sheldon ◽  
Matthew S. Macauley ◽  
Edward J. Taylor ◽  
Charlotte E. Robinson ◽  
Simon J. Charnock ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Group A Streptococcus ◽  
Competitive Inhibitor ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Toxic Shock ◽  
Nmr Studies ◽  
Invasive Infections ◽  
Group A

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called ‘flesh eating disease’) and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove β-O-linked N-acetylglucosamine from mammalian glycoproteins – an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure–reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2′-methyl-α-D-glucopyranoso-[2,1-d]-Δ2′-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic β-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases.

scholarly journals Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function

2020 ◽  
Vol 6 (10) ◽  
Author(s):  
Ao Li ◽  
Elisabeth Laville ◽  
Laurence Tarquis ◽  
Vincent Lombard ◽  
David Ropartz ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Sequence Similarity ◽  
Gut Bacteria ◽  
Glycoside Hydrolase Family ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Content Type ◽  
Multiple Sequence Alignments ◽  
Hydrolase Family

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

scholarly journals Characterization and Synergistic Interactions of Fibrobacter succinogenes Glycoside Hydrolases

2007 ◽  
Vol 73 (19) ◽  
pp. 6098-6105 ◽  
Author(s):  
Meng Qi ◽  
Hyun-Sik Jun ◽  
Cecil W. Forsberg
Keyword(s):  
Amino Acid ◽  
Synergistic Effect ◽  
Genome Sequence ◽  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Fibrobacter Succinogenes ◽  
Gene Encoding ◽  
Substrate Specificities ◽  
Synergistic Interactions

ABSTRACT The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

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

2000 ◽  
Vol 347 (3) ◽  
pp. 865-873 ◽  
Author(s):  
Patricia NTARIMA ◽  
Wim NERINCKX ◽  
Klaus KLARSKOV ◽  
Bart DEVREESE ◽  
Mahalingeshwara K. BHAT ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Thermomyces Lanuginosus ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
X Ray ◽  
Order Of Magnitude ◽  
Site Markers ◽  
Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

scholarly journals Characterization of Glycoside Hydrolase Families 13 and 31 Reveals Expansion and Diversification of α-Amylase Genes in the Phlebotomine Lutzomyia longipalpis and Modulation of Sandfly Glycosidase Activities by Leishmania Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Samara Graciane da Costa-Latgé ◽  
Paul Bates ◽  
Rod Dillon ◽  
Fernando Ariel Genta
Keyword(s):  
Glycoside Hydrolase ◽  
The Body ◽  
Glycoside Hydrolases ◽  
Food Sources ◽  
Leishmania Infection ◽  
Glycoside Hydrolase Family ◽  
Lutzomyia Longipalpis ◽  
Leishmania Mexicana ◽  
Hydrolase Family ◽  
Dipteran Species

Sugar-rich food sources are essential for sandflies to meet their energy demands, achieving more prolonged survival. The digestion of carbohydrates from food is mainly realized by glycoside hydrolases (GH). To identify genes coding for α-glycosidases and α-amylases belonging to Glycoside Hydrolase Family 13 (GH13) and Glycoside Hydrolase Family 31 (GH31) in Lutzomyia longipalpis, we performed an HMMER search against its genome using known sequences from other dipteran species. The sequences retrieved were classified based on BLASTP best hit, analysis of conserved regions by alignment with sequences of proteins with known structure, and phylogenetic analysis comparing with orthologous proteins from other dipteran species. Using RT-PCR analysis, we evaluated the expression of GH13 and GH31 genes, in the gut and rest of the body of females, in four different conditions: non-fed, sugar-fed, blood-fed, and Leishmania mexicana infected females. L. longipalpis has GH13/31 genes that code for enzymes involved in various aspects of sugar metabolism, as carbohydrate digestion, storage, and mobilization of glycogen reserves, proteins involved in transport, control of N-glycosylation quality, as well as others with a putative function in the regulation of myogenesis. These proteins are representatives of GH13 and GH31 families, and their roles seem to be conserved. Most of the enzymes seem to be active with conserved consense sequences, including the expected catalytic residues. α-amylases also demonstrated the presence of calcium and chloride binding sites. L. longipalpis genome shows an expansion in the α-amylase gene family, with two clusters. In contrast, a retraction in the number of α-glucosidases occurred. The expansion of α-amylases is probably related to the specialization of these proteins for different substrates or inhibitors, which might correlate with the higher diversity of plant foods available in the natural habitat of L. longipalpis. The expression of α-glucosidase genes is higher in blood-fed females, suggesting their role in blood digestion. Besides that, in blood-fed females infected with the parasite Leishmania mexicana, these genes were also modulated. Glycoside Hydrolases from families 13 and 31 are essential for the metabolism of L. longipalpis, and GH13 enzymes seem to be involved in the interaction between sandflies and Leishmania.

scholarly journals Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

2013 ◽  
Vol 288 (37) ◽  
pp. 26764-26774 ◽  
Author(s):  
Henriëtte J. Rozeboom ◽  
Shukun Yu ◽  
Susan Madrid ◽  
Kor H. Kalk ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Family Member ◽  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B14

2010 ◽  
Vol 192 (9) ◽  
pp. 2335-2345 ◽  
Author(s):  
Dylan Dodd ◽  
Shinichi Kiyonari ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Glycoside Hydrolase ◽  
Site Directed Mutagenesis ◽  
Rumen Bacterium ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
Sequence Alignments ◽  
Prevotella Bryantii

ABSTRACT Prevotella bryantii B14 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k cat/Km for pNP-β-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k cat/Km for pNP-β-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

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