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PubMed1988

Homology of glucosyltransferase gene and protein sequences from Streptococcus sobrinus and Streptococcus mutans.

The sequences of glucosyltransferase genes from Streptococcus sobrinus (gtfI) and Streptococcus mutans (gftB) were compared and show a high degree of homology. There is a 57.7% homology of nucleotides in the genes and a 56.7% homology of amino acids in the deduced protein sequences. The G + C content for the protein-coding region is 43.6% for S. sobrinus and 41.2% for S. mutans. Internal repeating sequences present in both proteins exhibit some difference in sequence pattern.

Amino Acid Sequence↗

PubMed1990

Characterization of monoclonal antibodies against glucosyltransferase synthesizing water-insoluble glucan from Streptococcus sobrinus B13.

Four hybrid cell lines secreting monoclonal antibodies (McAbs) against glucosyltransferase synthesizing water-insoluble glucan (GTase-I) were generated by fusion of myeloma cells (P3U1) with splenocytes from mice immunized with GTase-I from Streptococcus sobrinus B13-N. Cell lines 29E7, 21GC7, and 42HB8 were found to secrete an IgG2a-type immunoglobulin, and 29EG, an IgM-type immunoglobulin. These two isotypes of McAbs were used for the determination of the binding sites on the GTase molecule, with use of an enzyme-linked immunosorbent assay. The binding site for McAb 29EG was different from the site that bound other McAbs. McAb 29EG was found to inhibit water-insoluble glucan synthesis by GTase-I by 50%, and 29E7 was found to inhibit it weakly. However, other McAbs did not show any inhibitory effect in spite of binding to GTase-I. McAb 42HB8 strongly inhibited GTase-I-mediated adherence of heat-killed cells of S. sobrinus B-13N to glass surfaces. When the McAbs were tested for their cross-reactivity among GTase preparations from different mutans streptococci, McAb 29EG reacted with S. cricetus and S. sobrinus, but not with other mutans streptococci. Three other McAbs, 21GC7, 29E7, and 42HB8, were found to react only with the enzyme from S. sobrinus. These findings indicated that the specificity of the McAbs studied varied with respect to the antigenic sites on the GTase-I molecule, and that some of the sites differed in their functions.

Antibodies, Monoclonal↗

PubMed1994

Definition of a fundamental repeating unit in streptococcal glucosyltransferase glucan-binding regions and related sequences.

The C-termini of the glucosyltransferases (Gtfs) of oral streptococci are responsible for glucan binding. These glucan-binding domains (GBDs) are composed of a series of repeated sequences that have been classified into four different classes (A-D) by virtue of sequence similarity and which, by inference, have been suggested to be of functional importance. In contrast, we propose that repeat sequences evolve in response to selection for an increase in the number of copies of a particular domain through multiple duplication events occurring at different times. According to this hypothesis, repeats should possess various degrees of similarity, especially if only key residues are of functional importance. Analysis of the GBDs of the Gtfs indicated that a common fundamental repeat, designated the "YG" repeat, could be discerned within the "A", "B", "C", and "D" repeats. Similar elements were also conserved in the ligand-binding repeats of the Clostridium difficile toxins and the lysins and the PspA protein of Streptococcus pneumoniae, suggesting that similar selective pressures had also been imposed on these sequences. Analysis of the "YG" repeats present in the GtfJ and GtfK of Streptococcus salivarius indicated that some of the "YG" repeats in the GBDs of these proteins had arisen as a result of duplication events involving a series of three sequential "YG" repeats.

Amino Acid Sequence↗

PubMed2004

Construction and immunogenic characterization of a fusion anti-caries DNA vaccine against PAc and glucosyltransferase I of Streptococcus mutans.

Glucosyltransferases (GTFs) and A cell-surface protein (PAc) are two important virulence factors of the cariogenic organism Streptococcus mutans. They may mediate sucrose-independent or sucrose-dependent attachment of Streptococcus mutans to tooth surfaces, respectively. Thus, inhibiting both virulence factors is predicted to provide better protection against caries than inhibiting a single factor. To develop a highly efficient vaccine against caries, we constructed a fusion DNA vaccine, pGLUA-P, by cloning the GLU region of GTF into a DNA vaccine, pCIA-P, which encodes two highly conservative regions of PAc. In this report, we provide evidence that fewer caries lesions were observed in rats following subcutaneous injection of pGLUA-P, compared with pCIA-P, near the submandibular gland. Our findings suggest that a multigenic DNA vaccine may be more caries-preventive than a single-gene DNA vaccine.

Adhesins, Bacterial↗

PubMed2004

Synergistic effects of streptococcal glucosyltransferases on adhesive biofilm formation.

Glucosyltransferases (GTF)-I and GTF-SI of Streptococcus mutans synthesize water-insoluble and both water-soluble and -insoluble glucans, respectively, and play essential roles in the sucrose-dependent adhesion of the organism to tooth surfaces. To examine the interactions of different GTFs on artificial biofilm formed by S. mutans and other oral streptococci, we generated GTF-I- and GTF-SI-hyperproducing isogenic mutant strains. Transformant B42-21, which hyperexpressed GTF-SI, exhibited firm adhesion in the presence of sucrose, whereas transformant B42-10, which hyperexpressed GTF-I, failed to exhibit firm adhesion. Furthermore, co-culture of transformant B42-21 with water-soluble glucan-synthesizing Streptococcus sanguinis yielded firm adhesion, while the addition of dextran T10 to B42-21 growing culture had no effect on adhesion. These findings suggest that GTF-SI has a strong effect on sucrose-dependent adhesion and is essential for biofilm formation on smooth surfaces, in cooperation with water-soluble glucans synthesized de novo by oral streptococci that inherently lack cell adhesion ability.

Analysis of Variance↗

PubMed2003

Effect of a novel type of propolis and its chemical fractions on glucosyltransferases and on growth and adherence of mutans streptococci.

Flavonoids have been considered the main biologically active components in propolis. However, a new variety of flavonoid-free propolis was recently found and chemically classified as type 6. Because it showed activity against oral microorganisms, this study evaluated the effects of the crude ethanolic extract of this propolis and its chemical fractions on the activity of purified glucosyltransferases (GTFs) and on the growth and adherence of mutans streptococci. The inhibitory effect of propolis extracts on GTF activities was determined either in solution or adsorbed onto saliva-coated hydroxyapatite. Streptococcus mutans Ingbritt 1600, Streptococcus sobrinus 6715, and two clinical isolates of each species were used for antibacterial assays. Susceptibilities to the test extracts were analyzed using the agar diffusion method and by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC); the effect on bacterial adherence to a glass surface was also assessed. The activity of GTFs in solution was effectively inhibited by the ethanolic extract of propolis type 6 (EEP) (>80% inhibition at 0.5 mg/ml), hexane, and chloroform fractions (60-90% inhibition at 100 microg/ml); their inhibitory effects on surface enzymes were less pronounced. The EEP, hexane, and chloroform fractions also showed significant antibacterial activity. The data showed that propolis type 6 remarkably reduced GTF activity and inhibited mutans streptococci growth and adherence; these biological activities are associated with its nonpolar components.

Bacterial Adhesion↗

PubMed1990

Effect of tea polyphenols on glucan synthesis by glucosyltransferase from Streptococcus mutans.

In the course of our studies on the development of anti-plaque agents for prevention of dental caries, we investigated effects of some of tea preparations and their individual components on the glucan synthesis catalyzed by glucosyltransferase (GTF) from Streptococcus mutans. Extracts of green tea and black tea, and polyphenol mixtures showed appreciable inhibition in the synthesis of insoluble glucan. Among the components isolated from tea infusions, theaflavin and its mono- and digallates had potent inhibitory activities at concentrations of 1-10 mM against GTF. (+)-Catechin, (-)-epicatechin and their enantiomers had moderate inhibitory activities at these concentrations, while galloyl esters of (-)-epicatechin, (-)-epigallocatechin and (-)-gallocatechin had increased inhibitory activities.

Glucans↗

PubMed1994

Characterization of alpha-glucosyltransferase from Pseudomonas mesoacidophila MX-45.

alpha-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS-PAGE, and the isoelectric point was pI 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40 degrees C, respectively. The ranges of stable pH and temperature were pH 5.1-6.7 and below 40 degrees C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-alpha-D-glucopyranosyl- D-fructose) and isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5-6.5 at 20 degrees C. The yield of trehalulose from sucrose (20-40% solution) was 91%. The Km for sucrose was 19.2 +/- 3.3 mM estimated by the Hanes-Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 M. Hg2+, Fe3+, Cu2+, Mg2+, Ag+, Pb2+, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.

Bacterial Proteins↗

PubMed1995

Enzymatic formation of plant cerebroside: properties of UDP-glucose: ceramide glucosyltransferase in radish seedlings.

The activity of cerebroside synthase (UDP-glucose: ceramide glucosyltransferase) was found in the microsomal fraction of radish hypocotyls, and was studied. One % of the radioactivity due to UDP-[3H]glucose added to the membrane fraction was incorporated into cerebroside within 60 min. Optimum pH and temperature of the activity were pH 7.8 and 30 degrees C, respectively. No metal ions enhanced the cerebroside synthase activity, dissimilar to that in animal tissues. The apparent Km for UDP-glucose was approximately 200 microM. The same activity was also observed in radish roots and cotyledons, but proportions of UDP-[3H]glucose incorporation were slightly lower than that in hypocotyls. Exogenous ceramide species having trihydroxy sphingoid bases, which were major ceramide components of radish cerebrosides, usually stimulated cerebroside formation in microsomal fractions from radish seedlings, while any ceramide types having dihydroxy sphingoid bases were ineffective on the glucosylation reaction. It was assumed, therefore, that cerebroside synthase in radish seedlings would have substrate selectivity for ceramide species.

Brassica↗

PubMed2006

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana.

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.

Amino Acid Sequence↗

PubMed1999

Multi-enzymatic glucosylation using Eucalyptus UDP-glucosyltransferase coupled UDPglucose-fermentation by bakers' yeast.

The enzymatic synthesis of glucoside compounds using a membrane-associated UDP-glucosyltransferase fraction from Eucalyptus perriniana cultured cells as a water-insoluble catalyst (N. Nakajima, et. al., J. Ferment. Bioeng., 84 (5), pp. 455-460, 1997) has been effectively done by coupling UDPglucose-fermentation by bakers' yeast. For example, beta-thujaplicin (hinokitiol) and p-aminobenzoic acid were converted respectively to their corresponding beta-D-monoglucosides with the conversion rate of around 24-26% by the multi-enzymatic system with UDPglucose as a glucose donor, which is produced by yeast cells from glucose and 5'-UMP. Addition of either cellobiose, a substrate of beta-glucosidase, or DL-1,2-anhydro-myo-inositol, an inhibitor for the enzyme in the reaction mixture, could increased the yield of these beta-D-monoglucosides. This new enzymatic system could also be used for the synthesis of flavonoid glucosides such as isoquercitrin (quercetin 3-O-beta-D-glucoside).

Eucalyptus↗

PubMed2001

Purification and characterization of an oligo-isomaltosaccharide synthase from a Streptococcus sobrinus glucosyltransferase-I deficient mutant.

One glucosyltransferase (GTF) -I deficient mutant of Streptococcus sobrinus strain B13N was isolated through chemical mutagenesis with ethyl methanesulfonate, and characterized. This mutant, designated as B13N-Id, readily allowed us to purify a homogeneous oligo-isomaltosaccharide synthase (GTF-S) from its culture fluid. The purified GTF-S was only recognized with rabbit polyclonal antibody against recombinant GTF-S from an Ecsherichia coli MD124 clone expressing the B13N gtfS gene, and showed the almost same enzymatic properties as the recombinant enzyme. A double reciprocal plot of the B13N GTF-S for sucrose was biphasic, and the affinity for this substrate was high compared to that of GTF-S enzymes from other strains.

Chromatography, High Pressure Liquid↗

PubMed2005

Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans.

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.

Animals↗

PubMed2006

Isolation and characterization of anthocyanin 5-O-glucosyltransferase in Perilla frutescens var. crispa by differential display.

The glucosylation of 5-position of anthocyanin molecules is one of most important modification step, which is responsible for the stability and color hue of anthocyanin molecules. However, anthocyanin 5-O-glucosyltransferase (5-GT) is unstable and it had been difficult to purify this enzyme by biochemical methods. The isolation of cDNA encoding 5-GT was succeeded first by mRNA differential display (DD) of red and green chemotypes of Perilla frutescens var. crispa. The full-length cDNA was isolated by the screening of perilla cDNA library with cDNA fragments, which was displayed specifically in red perilla. The biological characters of this enzyme were investigated by using the recombinant protein expressed in yeast. We have shown an example of the application of DD technique to the molecular biology in plant secondary metabolisms.

Anthocyanins↗

PubMed2000

Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association.

Most glucosyltransferase (GTF) activity in sucrose-grown cultures of some strains of Leuconostoc mesenteroides is found with the cell pellet after centrifugation. GTFs are known to bind to dextrans, and it was traditionally assumed that cell-associated GTFs were bound to those dextrans that cosedimented with the cells. We used a mutant strain (LC-17), derived from strain NRRL B-1355, which produced dextransucrase in the absence of dextrans, to investigate the extent to which GTFs were bound to cells or dextrans. Much of the GTF activity in glucose-grown cultures of strain LC-17, which do not produce dextran, was located in the cell pellets. Soluble enzyme activity increased when cell suspensions from glucose- or sucrose-grown cultures were incubated with mild nonionic detergents or zwitterionic reagents. Alternansucrase produced by the parent strain B-1355 was almost entirely associated with cells under conditions in which dextrans were or were not produced. Alternansucrase, but not dextransucrase, tended to be enriched in the particulate fraction of B-1355 cells that had been broken in a French press. The distribution of alternansucrase and the effects of detergents on the distribution of GTFs suggest that soluble GTFs sequestered in the cytoplasm, and GTFs bound or adsorbed to the cell membrane are probably the major contributors to the cell-associated GTF activity.

Cell Membrane↗

PubMedDate not supplied

UDP-glucose: glucosyltransferase activity involved in the biosynthesis of flavonol triglucosides in Pisum sativum L. seedlings.

From young, light-grown seedlings of Pisum sativum L. an enzyme activity catalyzing the glucosylation of kaempferol and quercetin in the 3-position to form the 3-0-triglucoside derivative has been demonstrated. The reaction proceeds from the aglycone via the mono- and diglucoside intermediates. The triglucoside can be produced from any of the less substituted derivatives with uridine diphosphate-D-glucose (UDPG) as the glucosyl donor. Young leaf tissues had much higher levels of glucosyltransferase activity than the petioles and internodes. This is the first report of the synthesis of flavonol-3-0-triglucosides in vitro.

Flavonoids↗

PubMedDate not supplied

UDP-glucose: flavonol 7-O-glucosyltransferase activity in flower extracts of Chrysanthemum segetum.

The yellow colour of Chrysanthemum segetum petals is due to the presence of the 7-O-glucosides of quercetin and particularly gossypetin (8-hydroxyquercetin). In petal extracts of C. segetum an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5'-diphosphoglucose (UDPG) to the 7-hydroxyl group of flavonols with gossypetin and quercetin as the best substrates. Besides flavonols flavanones and flavones were found to be glucosylated in the 7-position. The pH-optimum of the reaction highly depended on the substrate used. With quercetin as substrate, maximal enzyme activity occurred at a pH of 8.25 and a temperature of 25 degrees C, but 7-O-glucosylation also proceeded at low temperatures. Studies on temperature stability revealed, that there was no influence on the glucosylation reaction up to 40 degrees C. Higher temperatures led to a loss of enzyme activity. Using gossypetin as a substrate a similar course of temperature stability was observed. Addition of Mg2+, Ca2+ and KCN slightly stimulated 7-O-glucosylation, whereas Co2+, Cu2+, Fe2+, Hg2+, p-hydroxymercuribenzoate and N-ethylmaleimide showed a strong inhibitory effect. Additional enzymatic studies were performed with the commercial strain "Stern des Orients" where gossypetin 7-O-glucoside is restricted to the inner parts of the petals. For enzyme extracts from both parts of the petals gossypetin was found to be the most attractive substrate. In comparison to quercetin (133.4 mu kat/kg protein) an about three times higher specific activity of the 7-O-glucosyltransferase(s) was determined with gossypetin (382.1 mu kat/kg protein) as substrate, indicating that hydroxylation of quercetin in 8-position to gossypetin precedes 7-O-glucosylation.

Chromatography, Gel↗

PubMedDate not supplied

Differential induction of glutathione transferases and glucosyltransferases in wheat, maize and Arabidopsis thaliana by herbicide safeners.

By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: (1) The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. (2) The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. (3) The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

Arabidopsis↗