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PubMed2005

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase.

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.

Bacillus↗

PubMed2004

Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells.

Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to various glucosides. The glucosylation efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures prior to curcumin administration. Two cDNAs encoding UDP-glucosyltransferases (CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C. roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases. The sequence identity between their deduced amino acid sequences was 27%. The expression of both genes was up-regulated by addition of MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than that of CaUGT2. The corresponding cDNAs were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant CaUGT1 exhibited no glucosylation activity with either curcumin or curcumin monoglucoside as substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin monoglucoside from curcumin and also conversion of curcumin monoglucoside to curcumin diglucoside. The use of the recombinant CaUGT2 may provide a useful new route for the production of curcumin glucosides.

Catalysis↗

PubMed2004

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutans.

Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.

Bacterial Proteins↗

PubMed2004

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system.

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.

Chromatography, Liquid↗

PubMed2002

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells.

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.

Amino Acid Sequence↗

PubMed1997

Production of glucosyltransferases by clinical mutans streptococcal isolates as determined by semiquantitative cross-dot assay.

Forty-four clinical isolates of mutans streptococci were examined by a semiquantitative cross-dot assay for in vitro production of glucosyltransferases GTF-I, GTF-SI and GTF-S of Streptococcus mutans, and GTF-I of Strep. sobrinus, using monospecific antibodies. The isolates were obtained from 12 1.5- to 3-year old children, six caries-active and six caries-free, and from their mothers. The isolates were selected originally from 243 isolates and they represented 35 genetically distinct types as analysed by serotyping and ribotyping. 27 isolates were of serotype c, nine of serotype e and eight of serotype g. Mother child pairs shared nine ribotypes, suggesting vertical transmission. The results showed that, when cultured in Todd-Hewitt broth supplemented with 1% glucose, all Strep. mutans isolates produced GTF-I and GTF-S and all except two produced GTF-SI of Strep. mutans. All Strep. sobrinus isolates produced GTF-I of Strep. sobrinus. The Strep. mutans GTF-I, GTF-SI and GTF-S production of isolates exhibiting a different ribotype showed variability. The variability of GTF-SI and GTF-S production was less pronounced for serotype e isolates. The GTF-I production by Strep. sobrinus isolates did not vary. Transmitted strains produced the same levels of GTFs as strains that were distinct (not transmitted). Strep. mutans isolates of caries-active children produced the same levels of GTF-I and GTF-S, but tended to produce lower levels of GTF-SI than isolates of caries-free children. In conclusion, the results suggested that Strep. mutans isolates exhibiting a different ribotype often had differences in production of GTFs. However, no clear superiority of the high-producer over the low-producer strains was found in regard to their colonization or caries promotion in young children.

Adult↗

PubMed1998

Binding properties of streptococcal glucosyltransferases for hydroxyapatite, saliva-coated hydroxyapatite, and bacterial surfaces.

The binding specificities of Streptococcus glucosyltransferase (Gtf) B, C and D for hydroxyapatite (HA), saliva-coated hydroxyapatite (SHA), and bacterial surfaces were examined. For HA beads the following values were obtained: (K = affinity; N = number of binding sites) GtfB, K = 46 x 10(5) ml/mumol, N = 0.65 x 10(-6) mumol/m2; GtfC, K = 86 x 10(5) ml/mumol, N = 4.42 x 10(-6) mumol/m2.; GtfD, K = 100 x 10(5) ml/mumol, N = 0.83 x 10(-6) mumol/m2. For SHA beads, the following values were obtained: GtfB, K = 14.7 x 10(5) ml/mumol, N = 1.03 x 10(-6) mumol/m2; GtfC, K = 21.3 x 10(5) ml/mumol, N = 3.66 x 10(-6) mumol/m2; GtfD, K = 1.73 x 10(5) ml/mumol, N = 8.88 x 10(-6) mumol/m2. The binding of GtfB to SHA beads was reduced in the presence of parotid saliva, but the binding of GtfC and D was unaffected. The binding of GtfB to SHA in the presence of parotid saliva supplemented with GtfC and D was reduced when compared with its binding to SHA in the presence of parotid saliva alone. In contrast, te binding of GtfC and SHA was unaffected when parotid saliva was supplemented with the other Gtf enzymes. GtfB bound to several bacterial strains (Strep, mutans GS-5, Actinomyces viscosus OMZ105E and Lactobacillus casei 4646) in an active form, while GtfC and D did not bind to bacterial surfaces. It is concluded that of the three Gtf enzymes, GtfC has the highest affinity for HA and SHA surfaces and can adsorb on the the SHA surface in the presence of the other two enzymes. GtfD also binds to SHA in the presence of the other enzymes but has a very low affinity for the surface. GtfB does not bind to SHA in the presence of the other Gtf enzymes but binds avidly to bacterial surfaces in an active form. Therefore, GtfC most probably binds to apatitic surfaces, while GtfB binds to bacterial surfaces.

Actinomyces viscosus↗

PubMed1998

Effects of surfactants on glucosyltransferase production and in vitro sucrose-dependent colonization by Streptococcus mutans.

The presence of Tween 80 in media was associated with a significant increase in three glucosyltransferases(GTFs)(I, SI and S), especially GTF-I, produced by Streptococcus mutans strain PS14, indicating that the surfactant is a major cause of the enhanced GTF production observed in cultures in M4 medium. Lecithin and Tween 20 also enhanced GTF-I production, while Triton X-100 depressed it. At a lot concentration of 0.00125%, Tween 80 enhanced markedly only GTF-I production and its effect reached maximum at a concentration of 0.0025%. Water-insoluble glucan synthesis and artificial plaque formation (in vitro sucrose-dependent colonization) by PS14 were significantly enhanced by the addition of Tween 80 at concentrations over 0.00125%. These results suggest that surfactants might vary the cariogenic potential of Strep. mutans even at low concentrations.

Bacterial Adhesion↗

PubMed1999

Action of agents on glucosyltransferases from Streptococcus mutans in solution and adsorbed to experimental pellicle.

Glucosyltransferase (Gtf) activity mediates sucrose-dependent adherence of mutans streptococci to the tooth surface, is essential for the cariogenicity of these micro-organisms, and contributes significantly to the exopolysaccharide component of the dental-plaque matrix. Clearly, agents that inhibit Gtfs could have therapeutic benefit. Here the effects of agents that inhibit Gtfs in solution and adsorbed to a surface were explored. Various classes of chemical reagents were tested for their ability to inhibit the enzymes responsible for insoluble-glucan synthesis (GtfB), insoluble/soluble glucan synthesis (GtfC), and soluble-glucan (GtfD) from Streptococcus mutans. Standard inhibition assays were done with Gtf enzyme in solution or with Gtf adsorbed to parotid saliva-coated hydroxylapatite (surface phase). Reagents tested included the metallic cations Li+, Zn2+, Cu2+, Fe2+ and Fe3+; the oxidizing compounds hypochlorite, Rose Bengal, perborate, and sodium-meta-periodate; and a panel of sugars and sugar analogues including sorbitol, xylitol, 1',4',6' trideoxy-trichloro-galactosucrose (TGS), and 1-deoxynojirimycin (dNJ). In solution, Gtf activity was inhibited significantly, at the highest concentrations tested: by the metal ions Zn2+, Cu2+, Fe2+ and Fe3+ (approx. 40-80% inhibition); by Rose Bengal and hypochlorite (approx. 80-90% inhibition); and by TGS and dNJ (approx. 50-80%). However, surface-adsorbed Gtfs displayed increased resistance to inhibition by the same metal cations and oxidizing compounds that inhibited them in solution. In contrast, both TGS and dNJ possessed similar inhibition profiles for both surface-bound Gtf and enzyme in solution. These data indicate that the nature of the inhibitor is important, and also whether the Gtf enzyme is in solution or adsorbed to saliva-coated hydroxylapatite.

1-Deoxynojirimycin↗

PubMed2000

Nigerooligosaccharide acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I.

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.

Bacterial Proteins↗

PubMed1981

D-Glucosyltransferase of Streptococcus mutans: isolation of two forms of the enzyme that bind to insoluble dextran.

The D-glucosyltransferase from Streptococcus mutans 6715 has been separated into three enzymic fractions that differ in their binding to dextran and in their synthesis of dextran from sucrose. One enzymic fraction (AFF-I) does not bind to insoluble dextran, and it produces an insoluble D-glucan. Fraction AFF-IIU was eluted from a dextran affinity-column by either dextran or urea, whereas fraction AFF-IID was eluted only by dextran. Both of these fractions produce insoluble D-glucans from sucrose.

Chromatography, Affinity↗

PubMed1986

Inhibition- and acceptor-reaction studies of Streptococcus mutans 6715 glucosyltransferases with 3-deoxysucrose, 3-deoxy-3-fluorosucrose, and alpha-D-allopyranosyl beta-D-fructofuranoside.

Three new sucrose analogs modified at C-3 have been studied as inhibitors and substrates for the glucosyltransferases (glucansucrases) of Streptococcus mutans 6715. Although none of the analogs were found to be substrates for polymer synthesis with either the soluble-polysaccharide producing enzyme, GTF-S, or the insoluble-polysaccharide producing enzyme, GTF-I, 3-deoxysucrose and 3-deoxy-3-fluorosucrose were able to donate glycosyl residues for acceptor reactions with both enzymes. Modification at C-3 considerably decreased the binding at the active site of both enzymes, since all of the analogs had inhibition constants at least one order of magnitude greater than the Km value for sucrose.

Chromatography, Thin Layer↗

PubMed2001

Dextran acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I as revealed by using uniformly 13C-labeled sucrose.

A sucrose glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus transferred the uniformly 13C-labeled glucosyl residue ([U-(13)C]Glc) from [U-(13)C]sucrose to exogenous dextran T500 at the non-reducing-end, mostly by alpha-(1-->6) linkages and partially by alpha-(1-->3) linkages, as revealed by the 13C-(13)C NMR coupling pattern. With increasing amounts of [U-(13)C]sucrose, transfer of [U-(13)C]Glc to the alpha-(1-->3)-linked chain became predominant without increase in the number of chains. The transfer of [U-(13)C]Glc to an isomaltopentaose acceptor occurred similarly to its transfer to T500. alpha-(1-->3)-branches in the [U-(13)C]dextran, specifically synthesized from [U-(13)C]sucrose by a Streptococcus bovis dextransucrase, were not formed by GTF-I, as judged by the observation that a newly-formed alpha-1,3,6-branched [U-(13)C]Glc was not detected, which could have been formed by transferring the unlabeled Glc from sucrose to the internal alpha-(1-->6)-linked [U-(13)C]Glc at C-3. The 13C-(13)C one-bond coupling constants (1J) were also recorded for the C-1--C-6 bond of the internal alpha-(1-->6)-linked [U-(13)C]Glc and of the non-reducing-end [U-(13)C]Glc.

Bacterial Proteins↗

PubMed1998

Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from [14C] maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase.

Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism. 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues. The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase. The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K. oxytoca M5a1.

Autoradiography↗

PubMed2000

Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus.

We isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Amino Acid Sequence↗

PubMed2000

Regulation of UDP-glucose:ceramide glucosyltransferase-1 by ceramide.

We report that the expression of mRNA and the activity of UDP-glucose:ceramide (Cer) glucosyltransferase-1 (GlcT-1) of human hepatoma Huh7 and mouse melanoma B16 cells increases after treatment with bacterial sphingomyelinase or upon addition of short-chain Cer. Interestingly, however, GlcT-1 gene transcription was not increased by Cer when GlcT-1 cDNA was introduced with the CMV promoter in GlcT-1-deficient GM95 cells, suggesting that the normal promoter region of GlcT-1 gene is essential for the response. The conversion of C6-Cer to C6-GlcCer occurred much more rapidly in GlcT-1-overexpressing Huh7 cells than in mock transfectants. As a result, GlcT-1-overexpressing cells acquired a greater resistance to C6-Cer-mediated cell death.

Animals↗

PubMed2001

Molecular cloning and disruption of a novel gene encoding UDP-glucose: tetrahydrobiopterin alpha-glucosyltransferase in the cyanobacterium Synechococcus sp. PCC 7942.

The gene encoding UDP-glucose:tetrahydrobiopterin alpha-glucosyltransferase (BGluT) was cloned from the genomic DNA of Synechococcus sp. PCC 7942. The encoded protein consisting of 359 amino acid residues was verified in vitro and in vivo to be responsible for the synthesis of tetrahydrobiopterin (BH4)-glucoside produced in the organism. The BGluT gene is the first cloned in pteridine glycosyltransferases and also a novel one cloned so far in UDP-glycosyltransferases. The mutant cells disrupted in the BGluT gene produced only aglycosidic BH4 at a level of 8.3% of the BH4-glucoside in wild type cells and exhibited half of the wild type growth in normal photoautotrophic conditions. These results suggest that the glucosylation of BH4 is required for the maintenance of the high cellular concentration of the compound, thereby supporting the normal growth of Synechococcus sp. PCC 7942.

Aclarubicin↗

PubMed1999

An early salicylic acid-, pathogen- and elicitor-inducible tobacco glucosyltransferase: role in compartmentalization of phenolics and H2O2 metabolism.

Treatment of tobacco cell suspension cultures with a fungal elicitor of defense responses resulted in an early accumulation of the phenylpropanoid glucosyltransferase TOGT, along with the rapid synthesis and secretion of scopolin, the glucoside of scopoletin. Elicitor-triggered extracellular accumulation of the aglycone scopoletin and of free caffeic and ferulic acids could only be revealed in the presence of diphenylene iodonium, an inhibitor of extracellular H2O2 production. Our results strongly support a role for TOGT in the elicitor-stimulated production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging.

Caffeic Acids↗