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At least 415 records · Page 23Linked to original sources
PubMed1998

Induction of KDNase Sm, a deaminoneuraminic acid (KDN) residue-specific sialidase from Sphingobacterium multivorum, using synthetic KDN-glycosides.

Various aryl and alkyl alpha-glycosides of KDN were synthesized and tested as substrates for their susceptibility to a deaminoneuraminic acid (KDN)-specific sialidase from Sphingobacterium multivorum, designated KDNase Sm. The synthetic KDN-glycosides were all hydrolyzed by the action of KDNase Sm. A hydroxyl group at C-5 position of KDN was required for the recognition by the enzyme, and was shown not to be replaced by an amino- or an acylamino group for the enzymatic recognition. These synthetic KDN-glycosides were also examined for their inducing activity of KDNase in S. multivorum and were shown to induce the KDNase activity effectively when the bacterium was cultured minimum salt medium containing both 0.1% glucose and 0.1% various KDN-glycosides. No KDNase activity was induced by the KDN-glycosides without 0.1% glucose. This is the first case of using synthetic KDN-glycosides as inducers of KDNase Sm.

Carbohydrate Conformation↗
PubMed1999

Crystal structure at 1.8 A resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus.

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P21 space group with a=41.7 A, b=68.1 A, c=51. 4 A and beta=113.6 degrees, Z=2 were grown that could diffract to better than 1.8 A resolution. The structure was solved by molecular replacement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multiple alignment of related xylanase sequences. The sequence thus obtained provides a correction to the sequence reported earlier based on biochemical methods. The final refined protein model at 1.8 A resolution with 301 amino acid residues and 266 water molecules has an R-factor of 16.0 % and free R of 21.1 % with good stereochemistry. The single polypeptide chain assumes (alpha/beta)8 TIM-barrel fold and belongs to F/10 family of glycoside hydrolases. The active site consists of two glutamate residues located at the C terminus end of the beta-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt bridges have been identified. In particular, the salt bridge Arg124-Glu232 which is almost buried, bridges the beta-strands beta4 and beta7 where the catalytic glutamate residues reside, and it may play a key role in the stability and activity at elevated temperature. To our knowledge, for the first time in the F/10 family xylanases, we observe a proline residue in the middle of the alpha-helix alpha6 which may be contributing to better packing. Earlier studies show that the enzyme retains its activity even at 70 degrees C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causative factors for thermostability are discussed.

Amino Acid Sequence↗
PubMed2000

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

Amino Acid Motifs↗
PubMed2000

Characterization of a membrane-associated protein implicated in visna virus binding and infection.

The identity of the cellular receptor(s) for visna virus, an ovine lentivirus, is currently unknown; however, previous studies from our laboratory have identified membrane-associated proteins expressed selectively in susceptible cells which bind visna virus. Moreover, a polyclonal antibody (2-23), raised against a 45-kDa visna virus binding protein, bound specifically to the surface of susceptible cells in immunofluorescence assays and significantly reduced binding of visna virus to cells (S. E. Crane et al., 1991, J. Virol., 65, 6137-6143). In this report we extend our studies of this antibody (2-23), showing both that 2-23 significantly reduces visna virus infection of susceptible cells and that 2-23 immunoprecipitates a putative protein complex consisting of a prominent 30-kDa protein, as well as the 45-kDa immunogen, specifically from radiolabeled virus-susceptible sheep cells. Further, we demonstrate that the 30-kDa protein is a membrane-associated proteoglycan substituted with a chondroitin sulfate glycosaminoglycan (GAG) chain(s) and that treatment of susceptible cells with an inhibitor of GAG synthesis significantly reduces visna virus production. Collectively, these data support a role for a proteoglycan in visna virus cell binding and infection.

Animals↗
PubMed2000

Characterization of a beta-1,3-glucanase encoded by chlorella virus PBCV-1.

Sequence analysis of the 330-kb chlorella virus PBCV-1 genome revealed an open-reading frame, A94L, that encodes a protein with significant amino acid identity to Glycoside Hydrolase Family 16 beta-1,3-glucanases. The a94l gene was cloned and the protein was expressed as a GST-A94L fusion protein in Escherichia coli. The recombinant A94L protein hydrolyzed the beta-1,3-glucose polymer laminarin and had slightly less hydrolytic activity on beta-1,3-1, 4-glucose polymers, lichenan and barley beta-glucan. The recombinant enzyme had the highest activity at 65 degrees C and pH 8. We predicted that the a94l-encoded beta-1,3-glucanase is involved in degrading the host cell wall either during virus release and/or is packaged in the virion particle and involved in virus entry. Therefore, we expected a94l to be expressed late in virus infection. However, contrary to expectations, both the a94l mRNA and the A94L protein appeared 15 min after PBCV-1 infection and disappeared 60- and 120-min p.i. postinfection, respectively, indicating that a94l is an early gene. Twenty-seven of 42 chlorella viruses contained the a94l gene. To our knowledge, this is the first report of a virus-encoded beta-1,3-glucanase.

Amino Acid Sequence↗
PubMed1978

A histochemical study of glycosidases in benign prostatic hyperplasia and in prostatic carcinoma in the human.

In benign hyperplasia marked beta-glucuronidase and N-acetyl-beta-glucosaminidase activity was seen in the prostatic epithelium whereas the reactions for 5 other glycosidases were only slight or negative. The intensity of the reaction for the glycosidases in prostatic carcinoma was dependent upon the degree of the differentiation. The possible importance of glycosidases for the invasiveness of prostatic carcinoma is briefly discussed.

Adenocarcinoma↗
PubMed1996

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl beta-D-lactoside derivatives.

4-Methylumbelliferyl 6'-O-benzyl-beta-D-lactoside (6'Bn-MU-Lac) and some related compounds were synthesized via different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6'Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, the K(m) value is 0.232 mM at pH 5.

Animals↗
PubMed1998

The isolation of genomic DNA from blackcurrant (Ribes nigrum L.).

A method is described for isolating DNA of high molecular mass (M(r)) from blackcurrant and other softfruit species. Following a hexacethylytimethyl ammonium bromide (CTAB)-based extraction procedure, samples are treated with a glycosidic hydrolase mixture and RNase, and then purified. The suitability of this DNA for Southern analysis and genomic-library construction is demonstrated.

DNA, Plant↗
PubMed2002

New endo-beta-1,4-glucanases from the parabasalian symbionts, Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites.

Abstract. An endo-beta-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli.

Amino Acid Sequence↗
PubMed2002

Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes formosanus.

Cellulase genes of Pseudotrichonympha grassii (Hypermastigida: Eucomonymphidae), the symbiotic flagellate in the hindgut of the wood-feeding termite Coptotermes formosanus, were isolated and characterized. The nucleotide sequences of the major cellulase component in the hindgut of C. formosanus were determined based on its N-terminal amino acid sequence. The five isolated nucleotide sequences (PgCBH-homos) had an open reading frame of 1350 bp showing similarity to catalytic domains of glycoside hydrolase family (GHF) 7 members, and primary structure comparison with GHF7 members whose tertiary structures are well-characterized revealed the overall similarity between PgCBH-homo and the catalytic domain of a processive cellulase Cel7A (formerly CBHI) from the aerobic fungus Trichoderma reesei. Functional expression of PgCBH-homos in Escherichia coli, using the carboxymethylcellulose-Congo red assay, demonstrated the actual cellulolytic activity of PgCBH-homo. RT-PCR showed that PgCBH-homos were expressed, from the three flagellates in the hindgut, specifically in P. grassii.

Amino Acid Sequence↗
PubMed2003

Solution structure and activity of mouse lysozyme M.

The three-dimensional structure of mouse lysozyme M, glycoside hydrolase, with 130 amino acids has been determined by heteronuclear NMR spectroscopy. We found that mouse lysozyme M had four alpha-helices, two 3(10)helices, and a double- and a triple-stranded anti-parallel beta-sheet, and its structure was very similar to that of hen lysozyme in solution and in the crystalline state. The pH activity profile of p-nitrophenyl penta N-acetyl-beta-D-chitopentaoside hydrolysis by mouse lysozyme M was similar to that of hen lysozyme, but the hydrolytic activity of mouse lysozyme M was lower. From analyses of binding affinities of lysozymes to a substrate analogue and internal motions of lysozymes, we suggest that the lower activity of mouse lysozyme M was due to the larger dissociation constant of its enzyme-substrate complex and the restricted internal backbone motions in the molecule.

Amino Acid Sequence↗
PubMed2005

Degradation of rice bran hemicellulose by Paenibacillus sp. strain HC1: gene cloning, characterization and function of beta-D-glucosidase as an enzyme involved in degradation.

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as beta-D-glucosidase and beta-D-arabinosidase. One of them, beta-D-glucosidase, was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for beta-D-glucosidase was cloned by screening for beta-D-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347 bp coding for a polypeptide with a molecular mass of 51.4 kDa. The polypeptide exhibits significant homology with other bacterial beta-D-glucosidases and belongs to glycoside hydrolase family 1. Beta-D-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50 kDa most active at around pH 7.0 and 37 degrees C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.

Amino Acid Sequence↗
PubMed2002

A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose.

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

Acetylglucosaminidase↗
PubMed2002

Molecular cloning, sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp PE2, and its domain structure.

The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.

Amino Acid Sequence↗