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PubMed1985

Isolation and characterization of an extracellular glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c.

A glucosyltransferase which synthesized insoluble glucan in polyacrylamide gel was isolated from the culture supernatant of Streptococcus mutans Ingbritt (serotype c) by ultrafiltration, ethanol fractionation, isoelectric focusing, and preparative gel electrophoresis. The enzyme preparation was electrophoretically homogeneous and immunologically distinct from the highly branched 1,6-alpha-D-glucan synthase and fructosyltransferase from the same strain and glucosyltransferases from serotypes a and g. The molecular weight was 99,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was 8.5. The enzyme had the optimum pH of 6.0 and Km value for sucrose of 9.4 mM. Besides the insoluble glucan with 96% 1,3-alpha linkage, this enzyme synthesized a considerable amount of diffusible glucan with 84% 1,6-alpha linkage, separately. This enzyme may be the one released from the enzyme aggregates by extracellular proteases, because the addition of extraneous trypsin to the crude enzyme preparation increased the amount of the enzyme species.

Animals↗

PubMed1988

Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus.

A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.

Amino Acids↗

PubMed1988

Sequence analysis of the glucosyltransferase A gene (gtfA) from Streptococcus mutans Ingbritt.

The complete nucleotide sequence of a 2.4-kilobase fragment containing the glucosyltransferase A gene (gtfA) of Streptococcus mutans Ingbritt (serotype c) was determined. The gtfA gene contains 481 codons and specifies a protein of molecular weight 55,665. There is no evidence of a signal peptide in the protein or that the glucosyltransferase A enzyme is secreted. No sequence homologies were observed between the gtfA gene or protein and the gtfI or gtfB gene and its protein.

Amino Acid Sequence↗

PubMed1990

Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide.

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.

Amino Acid Sequence↗

PubMed1991

Glucosyltransferase gene polymorphism among Streptococcus mutans strains.

Genetic polymorphisms in genes coding for the glucosyltransferases were detected among Streptococcus mutans serotype c strains by Southern blot analysis with DNA probes located within the gtfB gene (H. Aoki, T. Shiroza, M. Hayakawa, S. Sato, and H. K. Kuramitsu, Infect. Immun. 53:587-594, 1986). Restriction endonucleases were used to examine genomic DNAs isolated from serotype a to h strains. The variations were readily detected among 33 strains of serotype c by EcoRI and PstI restriction enzyme digestions. Serotypes e and f, which are genetically similar to serotype c, also had comparable polymorphism; however, serotypes a, b, d, g, and h did not hybridize to the same DNA probes in parallel experiments. Further analysis of enzymatic activities for glucan synthesis and sucrose-dependent adherence revealed no significant differences among the serotype c strains. Our results suggested that genetic polymorphisms existing in S. mutans serotype c strains may reflect a complexity in genes coding for the glucosyltransferases, which are produced ubiquitously in members of the S. mutans group.

Bacterial Adhesion↗

PubMed1991

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange.

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.

Alleles↗

PubMed1993

Inhibition of glucosyltransferase activities of Streptococcus mutans by a monoclonal antibody to a subsequence peptide.

Preliminary analysis indicated that a 19-amino-acid peptide sequence (435 to 453 of GtfC) within a highly conserved region of the glucosyltransferases of the cariogenic streptococci might be functionally important (J.-S. Chia, S.-W. Lin, T.-Y. Hsu, J.-Y. Chen, H.-W. Kwan, and C.-S. Yang, Infect. Immun. 61:1563-1566, 1993). To obtain antipeptide monoclonal antibodies (MAbs), the 19-amino-acid peptide was conjugated to bovine serum albumin and used as an antigen in BALB/c mice. Six immunoglobulin G-secreting hybridoma clones, CJSm18-S1 to -S6, specifically reacted with this peptide and with purified GtfC and GtfD but not with bovine serum albumin in an enzyme-linked immunosorbent assay. The concentrated hybridoma supernatant of all six MAbs inhibited GtfC enzymatic activity but failed to inhibit GtfD, although GtfD contains the same peptide sequence. Further analysis of a purified immunoglobulin G2b MAb from one of the clones, CJSm18-S3, confirmed that this MAb specifically inhibited GtfC enzymatic activity for insoluble-glucan synthesis in a dose-dependent manner. CJSm18-S3, even at high concentrations, had no effect on GtfD, which synthesizes water-soluble glucan exclusively. Furthermore, the in vitro sucrose-dependent adherence of Streptococcus mutans was also inhibited by CJSm18-S3 in a dose-dependent manner. Our results indicate that the peptide containing the N-terminal conserved region of glucosyltransferases is functionally important for both enzymatic activity and bacterial adherence.

Amino Acid Sequence↗

PubMed2000

Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and beta1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats.

To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

Amino Sugars↗

PubMed2006

Regulation of the glucosyltransferase (gtfBC) operon by CovR in Streptococcus mutans.

Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region.

Bacterial Proteins↗

PubMed1977

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Animals↗

PubMed2006

Glucosylation of flavonols by Escherichia coli expressing glucosyltransferase from rice (Oryza sativa).

A glucosyltransferase cDNA, RF5, was cloned from Oryza sativa using an RT-PCR strategy and expressed in Escherichia coli. Several flavonoids were tested for their ability to serve as substrates for RF5. RF5 effectively glucosylated kaempferol and quercetin to produce their 3-O-glucosides. Thus, RF5 could be defined as a flavonol 3-O-glucosyltransferase. E. coli cells expressing RF5 effectively converted 100 microM of kaempferol and quercetin into their corresponding glucosides.

Biotechnology↗

PubMed2002

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11.

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo [-->6]-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->)) from alpha-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-alpha-glucosyltransferase (6GT) catalyzing the a-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce alpha-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing alpha-1,3-, alpha-1,4-, and alpha,beta-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed alpha-1,3-transglycosylation, alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS was produced from alpha-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS-->alpha-isomaltosyl-->MOS (by 6GT), 2) alpha-isomaltosyl-MOS-->alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS (by IMT), and 3) alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS-->CTS + MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45 degrees C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40 degrees C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50 degrees C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40 degrees C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.

Bacillus↗

PubMed1975

Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

The glucosyltransferase (UDP-glucose galactosylhydroxylsine collagen glucosyltransferase, EC 2.4.1.?.) was purified 50-fold from calf arterial tissue by ammonium sulfate precipitation, gel filtration and electrofocusing. The purified enzyme has a molecular weight of 72 000 and a requirement for Mn2. It resolves into two activity peaks when submitted to electrofocusing (isoelectric point at pH 4.2 and 8.1) or disc electrophoresis and exhibits a double pH optimum (pH 8.3 and 9.9). The enzyme was found to transfer glucose from UDP-glucose to the denatured forms of citrate-soluble calf skin collagen (I), the alphal chain (II) and the beta12 component (III) derived from it, and of an acetic-acid-souble collagen preparation (IV) obtained from alkali-treated calf arterial tissue. The Km values for the substrates were 1.67 X 10(-4) (I), 6.3 X 10(-4) (II), 3.3 X 10(-4) (III) and 2.8 X 10(-4) mol/l (IV), indicating that the enzyme has the greatest affinity for the calf skin collagen. The glucose transferred to hydroxylysine-linked galactose residues may be released subsequently by the action of a specific alpha-glucosidase purified from bovine spleen. The results support the assumtion that the glucosylation step in the course of the (pro-)-collagen biosynthesis depends on special structural features of the substrate and may be controlled by a specific alpha-glucosidase.

Animals↗

PubMedDate not supplied

The ecdysteroid UDP-glucosyltransferase gene promoter from Autographa californica multicapsid nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (egt) gene promoter fragments of different lengths were generated from the genomic DNA of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) by PCR. After being purified and enzymatic digestion, they were cloned into the pGEM-3Z vector for construction of reporter plasmids pAcegt542-luc, pAcegt309-luc and pAcegt159-luc with the luciferase gene driven by the AcMNPV egt promoter. The results of transient expression in the Spodoptera frugiperda cell line-21 (Sf21) showed that the transcriptional activity of the AcMNPV egt promoter required the transactivation of viral factor(s). The expression of luciferase gene driven by the AcMNPV egt promoter was first detected at 24 h post infection. The egt promoter from the Bombyx mori nucleopolyhedrovirus (BmNPV), closely related to AcMNPV, revealed similar properties to that of the AcMNPV egt promoter. When BmNPV homologous region 3 was subcloned downstream the luciferase gene, the luciferase activity of the reporter plasmid was enhanced by over 1000-fold, but the property of the promoter was not changed. As a substrate of ecdysteroid UDP-glucosyltransferase (EGT), foreign insect ecdysone showed negative effects on egt promoter transcriptional activity. Ecdysone of 1.0-2.0 microg/ml significantly down-regulated the promoter activity. Promoter activities of different lengths showed that an AcMNPV egt promoter fragment of 159 bp has the basal transcriptional activity but it was almost abolished only about 0.2% of that of 309 bp and 542 bp, respectively, and the nucleotide sequence from - 159 to - 309 nt upstream the translation initiation site includes the main cis-acting elements interacting with viral factors.

Animals↗

PubMed1987

Inhibition of Streptococcus mutans glucosyltransferase by M-GTFI, a new inhibitor.

Two hundred strains of soil microorganisms were screened for the production of inhibitors of the glucosyltransferase activity of Streptococcus mutans strain, K1-R. The strain producing the greatest amount of inhibitor was one recently isolated in our laboratory. It has now been identified as a strain of Micromonospora narashinoensis on the basis of morphological and physiological studies. The inhibitor, M-GTFI, affects the glucosyltransferase that produces the water-insoluble glucan rather than that which produces the water-soluble glucan. Fuchsin-sulphite staining of the inhibitor after its purification by polyacrylamide gel electrophoresis indicates that it is probably an acidic substance. It had Mr 5700 as was determined by gel filtration. From an examination of the effects of this inhibitor on representative strains of S. mutans other than K1-R, there is a suggestion of a similar selectivity for the water-insoluble glucan-forming activity in other strains.

Dental Caries↗

PubMed1982

Specificity of a particulate glucosyltransferase in seedlings of Pisum sativum L. which catalyzes the formation of 5'-O-(beta-D-glucopyranosyl)pyridoxine.

A glucosyltransferase, catalyzing the transfer of D-glucose from UDP-glucose to the 5'-hydroxyl group of pyridoxine, was isolated as a particulate enzyme from seedlings of podded pea (Pisum sativum L. cv. Kinusaya). The enzyme required additional Mg2+ for its function. The pH optimum for glucosylation of pyridoxine was between 7.8 and 8.8. The enzyme showed high specificity for UDP-glucose and relative specificity for glucosyl acceptor: pyridoxine was replaceable by pyridoxamine. Several compounds tested other than vitamin B6 failed to serve as the acceptor. It was shown that a methylene group on C-4 participated in the formation of enzyme-substrate complex, and that the rate of glucosylation was dependent upon the C-4 substituent. From the results of kinetic studies and an experiment in vivo, the enzyme was inferred to be UDP-glucose: pyridoxine 5'-O-beta-glucosyltransferase.

Fabaceae↗

PubMed2000

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms.

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.

Candida albicans↗

PubMed1990

Biosynthesis of xanthurenic acid 8-O-beta-D-glucoside in Drosophila. Characterization of the xanthurenic acid:UDP-glucosyltransferase activity.

Xanthurenic acid 8-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila. From 3-hydroxykynurenine, two biosynthetic pathways can be envisaged, one via xanthurenic acid, and another via 3-O-glucoside of 3-hydroxykynurenine. In this report evidence is presented to show that the synthesis takes place via xanthurenic acid. (a) We have demonstrated that the Drosophila melanogaster vermilion purple mutant (unable to synthesize 3-hydroxykynurenine) synthesizes xanthurenic acid 8-glucoside when fed with xanthurenic acid; and (b) the activities required for its synthesis via xanthurenic acid have been found (3-hydroxykynurenine transaminase and xanthurenic acid:UDP-glucosyltransferase). This is the first time that a UDP-glucosyltransferase activity that utilizes xanthurenic acid has been demonstrated. The enzyme in crude extracts from Drosophila sordidula shows the following characteristics. (a) It has optimal activity at 35 degrees C at pH 7.1 (in buffer Tris-HCl), and in the presence of a divalent cation (Mg2+ or Mn2+); (b) the activity is inhibited by xanthurenic acid (above 1.5 mM), UDP, D-gluconic acid 1,5-lactone, and Triton X-100; (c) it is localized in both the microsomal and the soluble fractions; (d) the specific activity is two times higher in heads than in bodies; and (e) the activity is enhanced in flies fed with phenobarbital.

Animals↗