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 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 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 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 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 A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

scholarly journals Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Jennifer Hiras ◽  
Yu-Wei Wu ◽  
Kai Deng ◽  
Carrie D. Nicora ◽  
Joshua T. Aldrich ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Glycoside Hydrolase ◽  
Plant Biomass ◽  
Cellulose Hydrolysis ◽  
Global Carbon Cycle ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Global Carbon ◽  
Hydrolase Family

ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora , the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCE Cellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.

scholarly journals Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

2016 ◽  
Author(s):  
Jiujun Cheng ◽  
Tatyana Romantsov ◽  
Katja Engel ◽  
Andrew C. Doxey ◽  
David R. Rose ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Sinorhizobium Meliloti ◽  
Biochemical Analysis ◽  
Sequence Similarity ◽  
Metagenomic Library ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Galactosidase Activity ◽  
Functional Metagenomics ◽  
Hydrolase Family

AbstractA soil metagenomic library carried in pJC8 (an IncP cosmid) was used for functional complementation for β-galactosidase activity in bothα-Proteobacteria (Sinorhizobium meliloti)andγ-Proteobacteria (Escherichia coli).Oneβ-galactosidase, encoded by overlapping clones selected in both hosts, was identified as a member of glycoside hydrolase family 2. ORFs obviously encoding possible β-galactosidases were not identified in 19 other clones that were only able to complementS. meliloti.Based on low sequence similarity to known glycoside hydrolases but not β-galactosidases, three ORFs were examined further. Biochemical analysis confirmed that all encodedβ-galactosidase activity. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain.

scholarly journals Novel Members of Glycoside Hydrolase Family 13 Derived from Environmental DNA

2008 ◽  
Vol 74 (6) ◽  
pp. 1914-1921 ◽  
Author(s):  
Antje Labes ◽  
Eva Nordberg Karlsson ◽  
Olafur H. Fridjonsson ◽  
Pernilla Turner ◽  
Gudmundur O. Hreggvidson ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Environmental Dna ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Biotechnological Applications ◽  
Consensus Primer ◽  
Thermophilic Conditions ◽  
Maltogenic Amylase ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

ABSTRACT Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications.

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