scholarly journals Purification, characterization and gene cloning of two α-l-arabinofuranosidases from Streptomyces chartreusis GS901

2000 ◽  
Vol 346 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Noriki MATSUO ◽  
Satoshi KANEKO ◽  
Atsushi KUNO ◽  
Hideyuki KOBAYASHI ◽  
Isao KUSAKABE
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Gum Arabic ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Sequencing ◽  
Molecular Masses ◽  
Hydrolase Family

α-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. α-L-Arabinofuranosidase I hydrolysed all of the α-linkage types that normally occur between two α-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: α-(1 → 2) α-(1 → 3) α-(1 → 5). This enzyme cleaved the (1 → 3) linkages of the arabinosyl side-chains of methyl 3,5-di-O-α-L-arabinofuranosyl-α-L-arabinofuranoside in preference to the (1 → 5) linkages. α-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, α-L-Arabinofuranosidase II hydrolysed only (1 → 5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1 → 5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of α-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of α-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1,5-α-L-arabinofuranosidase, it represents a novel type of enzyme.

scholarly journals Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family

2011 ◽  
Vol 77 (23) ◽  
pp. 8350-8354 ◽  
Author(s):  
Yuichi Sakamoto ◽  
Keiko Nakade ◽  
Naotake Konno
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Fruiting Body ◽  
Glycoside Hydrolase ◽  
Lentinula Edodes ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Product Analysis ◽  
Content Type ◽  
Hydrolase Family

ABSTRACTThe cell wall of the fruiting body of the mushroomLentinula edodesis degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified fromL. edodesfruiting bodies after harvesting. The gene encoding it,glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed inPichia pastorisexhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).

scholarly journals An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2

2002 ◽  
Vol 365 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter J.M. STEENBAKKERS ◽  
Wimal UBHAYASEKERA ◽  
Harry J.A.M. GOOSSEN ◽  
Erik M.H.M. van LIEROP ◽  
Chris van der DRIFT ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Large Family ◽  
Glycoside Hydrolase Family ◽  
Reading Frame ◽  
Glycoside Hydrolase Family 9 ◽  
Molecular Masses ◽  
Β Sheet ◽  
Hydrolase Family

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170kDa components. To investigate the major 90kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800bp cDNA clone contained an open reading frame of 2334bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative β-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

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.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

scholarly journals Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

2006 ◽  
Vol 72 (9) ◽  
pp. 6399-6401 ◽  
Author(s):  
Jinhua Dong ◽  
Shinnosuke Hashikawa ◽  
Takafumi Konishi ◽  
Yutaka Tamaru ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Gene Product ◽  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hydrolase Family

ABSTRACT The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

scholarly journals Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11

2002 ◽  
Vol 184 (15) ◽  
pp. 4124-4133 ◽  
Author(s):  
Kaveh Emami ◽  
Tibor Nagy ◽  
Carlos M. G. A. Fontes ◽  
Luis M. A. Ferreira ◽  
Harry J. Gilbert
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Xylanase Activity ◽  
Glycoside Hydrolase Family ◽  
Side Chains ◽  
Temporal Regulation ◽  
Degrading Bacterium ◽  
Genes Encoding ◽  
Combined Action ◽  
Hydrolase Family

ABSTRACT Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

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.

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