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PubMed2004

Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas.

Saffron, the dry stigma of Crocus sativus L., is considered to be the world's most expensive spice. Three major apocarotenoids--crocin, crocetin and picrocrocin--are responsible for the colour and bitter taste of saffron. The final step in the biosynthesis of the 20-carbon esterified carotenoid crocin is the transformation of the insoluble crocetin into a soluble and stable storage form by glucosylation. These glucosylation reactions are catalysed by glucosyltransferases (GTases) that play a crucial role in natural-product biosynthesis. Using degenerate primers designed to match the plant secondary product GTase (PSPG) box we cloned two cDNAs, UGTCs2 and UGTCs3, from C. sativus stigmas that encode putative polypeptides of 460 and 475 amino acids, respectively. These genes were expressed differentially in saffron tissues. UGTCs2 was mainly expressed in fully developed stigmas, whereas UGTCs3 was mainly expressed in stamens. The UGTCs2 transcript was not detected in the stigma tissue of a Crocus species that does not synthesize crocin, while UGTCs3 and other structural genes for carotenoid biosynthesis were expressed in the stigma of all tested Crocus species. To identify the biochemical function of UGTCs2, the isolated cDNA was expressed in Escherichia coli cells. The recombinant protein UGTCs2 had glucosylation activity against crocetin, crocetin beta-D-glucosyl ester and crocetin beta-D-gentibiosyl ester. These results might suggest that the isolated clone UGTCs2 codes for a saffron crocetin GTase.

Amino Acid Sequence↗

PubMed2005

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (Oryza sativa L.) by suppression subtractive hybridization.

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5'- and 3'-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17-1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970-20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293-51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.

Amino Acid Sequence↗

PubMed1991

UDP-glucosyltransferase activity toward exogenous substrates in Drosophila melanogaster.

To investigate the capacity of Drosophila extracts to glucosylate exogenous substrates we have developed a fast and sensitive method for the detection of UDP-glucosyltransferase activity using 4-nitrophenol, 1-naphthol, or 2-naphthol as substrates. High-performance liquid chromatography was used to separate and quantitate the reaction products, allowing detection of activities that produced as little as 1 pmol of 2-naphthol glucoside (fluorescence detection) or 16 pmol of 4-nitrophenol glucoside (absorbance detection). Optimal activity was found at 43 degrees C and alkaline pH. The affinity of the Drosophila enzyme was 250-fold higher for 1-naphthol or 2-naphthol (Km approximately 4 microM) than for 4-nitrophenol and UDP-glucose (Km approximately 1 mM).

Animals↗

PubMed1988

A biochemical chimera suggesting the existence of at least two dolichol-P-glucose:dolichol-P-P-oligosaccharide glucosyltransferases.

As previously reported, incubation of liver dolichol-P, UDP-[14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[14C]Gal (P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205-212). It is now reported that upon incubation of the latter with rat liver microsomes, [14C-galactose]-Gal1Man9GlcNAc2-P-P-dolichol and [14C-galactose]Gal1Glc1Man9GlcNAc2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal1Glc2Man9GlcNAc2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.

Acetylation↗

PubMed1983

Inhibition by acarbose, nojirimycin and 1-deoxynojirimycin of glucosyltransferase produced by oral streptococci.

Acarbose is known to inhibit glucoamylase, maltase and sucrase. Our aim was to test whether it would also inhibit glucosyltransferase (GTF), to determine the type of inhibition and to compare the inhibitor potency of acarbose with that of nojirimycin and deoxynojirimycin, two other glucosidase inhibitors. Enzyme inhibition was measured either by chemical assay or by incorporation of radioactivity into product. Acarbose effectively inhibited the synthesis of polysaccharide by GTF from strains of Streptococcus mutans and Streptococcus sanguis, but not by fructosyltransferase from Streptococcus salivarius. Acarbose and 1-deoxynojirimycin were more potent inhibitors of GTF than maltose, nojirimycin or various amino sugars. The mechanism of action of these compounds is consistent with competitive inhibition.

1-Deoxynojirimycin↗

PubMed1985

Salivary-IgA antibody to glucosyltransferase of oral microbial origin in children.

Salivary IgA levels were measured in whole saliva of 143 children, 2-48 months of age. The median salivary IgA concentration of children less than 12 months old was 30 micrograms/ml. A significant increase in salivary IgA concentration occurred in children 12 to 17 months old. More than 50 per cent of the population, 36 months of age and older, had Streptococcus mutans (88 per cent biotype I) recoverable from their teeth. Salivary IgA antibody levels were measured by an enzyme-linked immunosorbent assay to glucosyltransferase (GTF) antigen complexes from oral streptococci. Salivary IgA antibody to GTF from Streptococcus sanguis was detected in many children. Over 40 per cent of the 43-48-month-old group had detectable salivary antibody to Strep. sanguis GTF; salivary IgA antibody to Strep. mutans GTF (biotype I) was less frequently detected. Most subjects with salivary antibody to Strep. mutans GTF had antibody to Strep. sanguis GTF but only 23 per cent with salivary antibody to Strep. sanguis GTF had antibody to Strep. mutans GTF. Antibody to Strep. sanguis GTF was infrequently associated with the recovery of Strep. mutans from the teeth.

Age Factors↗

PubMed1996

Interactions of streptococcal glucosyltransferases with alpha-amylase and starch on the surface of saliva-coated hydroxyapatite.

The salivary pellicle consists of various proteins and glycoproteins which may interact with one another. Experiments were performed to elucidate the interactions of streptococcal glucosyltransferase (Gtf) enzymes with human salivary alpha-amylase in solution and on the surface of saliva-coated hydroxyapatite (SHA) beads. The Gtf enzymes -B, -C and -D, when immobilized on to SHA beads, reduced the activity of adsorbed amylase; GtfD showed the highest inhibition of salivary amylase activity. The presence of glucan produced by immobilized GtfD did not further reduce amylase activity. The amount of amylase adsorbed on to hydroxyapatite beads was reduced when salivary amylase was added simultaneously with any of the Gtf enzymes, suggesting that amylase and Gtfs may compete with each other for binding sites on hydroxyapatite. Starch hydrolysates produced by SHA-surface-bound salivary amylase were tested for their effect on glucan production from sucrose by Gtf enzymes in solution and on SHA beads; glucan production by SHA-immobilized GtfB was stimulated in the presence of starch hydrolysates. Glucan synthesized by SHA-immobilized GtfB in the presence of starch hydrolysates was less susceptible to hydrolysis by the fungal enzyme mutanase than was glucan made by SHA-immobilized GtfB in the absence of starch hydrolysates. Glucan production by GtfB associated with streptococci immobilized on to SHA was also enhanced in the presence of starch hydrolysates. The adhesion of oral micro-organisms to SHA coated with glucan made in the presence and absence of starch hydrolysates was investigated, and some bacteria displayed higher adhesion activities for the glucan made in the presence of the hydrolysates. Therefore, the interaction of amylase and Gtf enzymes on a SHA surface may modulate the formation of glucan and the adherence of oral micro-organisms.

Adsorption↗

PubMed1977

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos.

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.

Animals↗

PubMed1992

Molecular characterization and expression of the cell-associated glucosyltransferase gene from Streptococcus mutans.

A gene encoding cell-associated glucosyltransferase (CA-GTase) was cloned from Streptococcus mutans MT8148 into Escherichia coli DH5 alpha by using a low-copy-number plasmid, pMW119. After screening of a gene library with the oligonucleotide probe designed on the basis of a partial amino acid sequence of CA-GTase, a recombinant plasmid, pSK6, that had a 5.6 kb insert carrying the CA-GTase gene was selected. The gene product (recombinant CA-GTase) of pSK6 was expressed by using a lac promoter in pMW119. Western blotting revealed that rCA-GTase reacted with antibody to CA-GTase. rCA-GTase was found to synthesize water-insoluble glucans. Southern blotting indicated that the MT8148 chromosome contained another gene which was homologous to pSK6. A plasmid harboring this gene (pSK16) was also isolated from the gene library, the gene product of pSK16 exhibited GTase activity but ten times lower than that of pSK6.

Base Sequence↗

PubMed1992

Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases.

In the present communication molecular genetic approaches have been utilized to confirm the nature of the catalytic site of Streptococcus mutans glucosyltransferases (GTF)s. Site-directed mutagenesis was used to convert the putative sucrose binding Asp-451 of the GTF-I enzyme from S. mutans GS5 to Glu, Asn, and Thr. All three of the resulting mutated enzymes displayed no detectable sucrase or GTF activities. By contrast, mutation of nearby Asp residues did not markedly reduce enzymatic activity. The inactive enzymes also appear to bind acceptor dextrans as well as the parental enzyme. These results confirm the essential role of Asp-451 of the GTF-I from strain GS5 and analogous Asp residues in other related GTFs in enzymatic activity.

Amino Acid Sequence↗

PubMed1984

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715.

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the "specific" binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependent GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.

Chromatography, Affinity↗

PubMed1985

Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6'.

Sucrose derivatives modified at position 6 (6-deoxysucrose, 6-thiosucrose, 6,6'-dithiodisucrose, and 6,6'-dideoxy-6,6'-difluorosucrose) were tested as inhibitors of the two Streptococcus mutans 6715 glucosyltransferases. 6-Deoxysucrose was the best inhibitor studied, competitively inhibiting the soluble-D-glucan forming enzyme (GTF-S) and the insoluble-D-glucan forming enzyme (GTF-I) with Ki values one order of magnitude lower than the sucrose Km values. 6-Thiosucrose was also a competitive inhibitor for both enzymes. 6,6'-Dithiodisucrose and 6,6'-dideoxy-6,6'-difluorosucrose only inhibited GTF-I; 6,6'-dithiodisucrose gave mixed inhibition and 6,6'-dideoxy-6,6'-difluorosucrose gave uncompetitive inhibition. 6-Thiosucrose was a substrate for both enzymes to produce acceptor products when acceptors were present. GTF-I synthesized de novo a water-insoluble, (1----3)-6-thio-alpha-D-glucan from 6-thiosucrose.

Binding, Competitive↗

PubMed1986

Purification and characterisation of the extracellular D-glucosyltransferase from serotype c Streptococcus mutans.

A simple method of purification for the extracellular D-glucosyltransferase (GTase) from a serotype c strain Streptococcus mutans was developed using chromatography on DEAE-Sephacel and CM-cellulose. The GTase had a molecular weight of 155,000 and an isoelectric point of 7.4. The enzyme converted sucrose, in the absence of dextran T-10, into a branched (1----6)-linked alpha-D-glucan having some alpha-(1----3)-linked D-glucosyl residues. The GTase was similar to GTases which have been isolated from other strains of serotype c S. mutans and which synthesise water-soluble glucans. In addition, the amino acid composition of the GTase protein was relatively similar to those of the GTases from serotype g S. mutans which synthesise water-soluble and water-insoluble glucans.

Amino Acids↗

PubMed1986

Difference in mode of inhibition between alpha-D-xylosyl beta-D-fructoside and alpha-isomaltosyl beta-D-fructoside in synthesis of glucan by Streptococcus mutans D-glucosyltransferase.

Both alpha-isomaltosyl beta-D-fructoside and alpha-D-xylosyl beta-D-fructoside show strong inhibition of the synthesis of water-insoluble and water-soluble D-glucans from sucrose by a partially purified preparation of a D-glucosyltransferase (GTase) from Streptococcus mutans 6715; however, the inhibitory modes differ substantially. In the presence of alpha-isomaltosyl beta-D-fructoside, the production of reducing sugars and the consumption of sucrose are remarkably enhanced, compared with a control of sucrose alone. Under these conditions, a large proportion of low-molecular-weight glycan (lmwg) and a series of nonreducing oligosaccharides (both containing D-fructosyl groups or residues) are produced. In contrast, in the presence of alpha-D-xylosyl beta-D-fructoside, the production of reducing sugars and the sucrose consumption are strikingly suppressed, and no lmwg or oligosaccharides are produced. Thus, it may be concluded that alpha-isomaltosyl beta-D-fructoside acts as an alternative acceptor for the D-glucosyl and/or D-glucanosyl transfer reactions of the enzyme, and serves to lessen the formation of insoluble and soluble D-glucan, although it stimulates the transferring activity of the enzyme. On the other hand, alpha-D-xylosyl beta-D-fructoside competitively inhibits the sucrose-splitting activity of the enzyme as an analog to sucrose, and thereby diminishes the synthesis of D-glucan.

Disaccharides↗

PubMed1986

Activity of branched dextrans in the acceptor reaction of a glucosyltransferase (GTF-I) from Streptococcus mutans OMZ176.

The ability of several native and chemically synthesized, branched dextrans to stimulate the activity of an alpha-D-glucosyltransferase (GTF-I) of Streptococcus mutans has been compared. The enzyme catalysed the transfer of glucosyl residues from sucrose with the formation of water-insoluble (1----3)-alpha-D-glucan. The rate of this reaction was greatly increased in the presence of dextran, and the extent of stimulation was negatively correlated with the degree of branching of the added dextran. The results refute the concept that growth of water-insoluble glucan occurs from the multiple, non-reducing termini of dextran acceptors.

Carbon Radioisotopes↗

PubMed1989

Inhibition by maltose, isomaltose, and nigerose of the synthesis of high-molecular-weight D-glucans by the D-glucosyltransferases of Streptococcus sobrinus.

Two D-glucosyltransferases are produced by Streptococcus sobrinus C211. One (GTF-S) catalyzes the conversion of sucrose into soluble alpha-(1----6)-linked alpha-(1----3)-branched D-glucans, and the other (GTF-I), of sucrose into alpha-(1----3)-linked alpha-(1----6)-branched D-glucans. These enzymes were studied by using maltose, isomaltose, and nigerose as inhibitors. Maltose and isomaltose were found to be competitive inhibitors of GTF-S, whereas nigerose has no effect on GTF-S activity. The Ki values for maltose and isomaltose were determined to be 11 and 15mM, respectively. Maltose, isomaltose, and nigerose competitively inhibit GTF-I. The Ki values for these inhibitors were found to be approximately 0.8, 2.5, and 15mM, respectively. The inhibitory properties of each disaccharide are interpreted in terms of conformational comparisons with sucrose.

Carbohydrate Conformation↗

PubMed1991

Maltodextrin acceptor reactions of Streptococcus mutans 6715 glucosyltransferases.

The maltodextrin (maltose through maltoheptaose) acceptor reactions of two Streptococcus mutans 6715 glucosyltransferases (GTF-I and GTF-S) were studied. The acceptor product structures were determined by comparing them with the known structures of the acceptor products of Leuconostoc mesenteroides B-512FM dextransucrase (EC 2.4.1.5) and L. mesenteroides B-1355 alternansucrase (EC 2.4.1.140). When reacted with maltose (G2), both GTF-I and GTF-S transferred a D-glucopyranose from sucrose to the nonreducing glucosyl residue to give panose (6(2)-alpha-D-glucopyranosyl maltose). Panose then served as an acceptor to give two further acceptor products, 6(2)-alpha-isomaltosyl maltose and 6(2)-alpha-nigerosyl maltose. 6(2)-alpha-Isomaltosyl maltose then went on to serve as an acceptor to give a series of homologous acceptor products with isomaltodextrin chains attached to C-6 of the nonreducing-end residue of maltose, while 6(2)-alpha-nigerosyl maltose did not further react. When reacted with other maltodextrins (G3-G7), both GTF-I and GTF-S transferred a D-glucopyranose to C-6 of either the nonreducing-end or the reducing-end residues of the maltodextrins, forming alpha(1----6) linkages. When D-glucopyranose was transferred to the nonreducing-end residue by GTF-I or GTF-S, the first product was also an acceptor to give the second product, which then served as an acceptor to give the third product, etc., to give a homologous series of products. When D-glucopyranose was transferred to the reducing-end residue, the acceptor product that formed did not readily serve as an acceptor, or served only as a very poor acceptor, to give a small amount of the next homologue, as was the case for G7 with GTF-S. In addition, GTF-I also transferred D-glucopyranose to the reducing-end or to the nonreducing-end residue of maltotriose, forming alpha(1----3) linkages, to give 3(3)-alpha-D-glucopyranosyl maltotriose and 3(1)-alpha-D-glucopyranosyl maltotriose. Neither of these acceptor products further served as acceptors to give a homologous series. Under equivalent conditions of equimolar amounts of acceptor and sucrose, maltose and maltotriose are much better acceptors with GTF-I than they are with GTF-S, which is better than L. mesenteroides B-512FM dextransucrase. The three enzymes display significantly different efficiencies for the different maltodextrin acceptor reactions, GTF-I and GTF-S having much higher efficiencies than L. mesenteroides B-512FM dextransucrase.

Carbohydrate Conformation↗

PubMed1996

Synthesis of 1',6'-disubstituted sucroses and their behavior as glucosyl donors for a microbial alpha-glucosyltransferase.

Versatile 6'-chloro-6'-deoxy-1'-substituted sucrose derivatives were synthesized in search of an optimum donor substrate for the intermolecular transglucosylation with the alpha-glucosyltransferase from Protaminobacter rubrum. Two substituents at the C-1' and C-6' positions of sucrose were introduced utilizing the distinct reactivity of the corresponding sulfonates. Methyl beta-D-arabinofuranoside was most efficiently glucosylated with the 1'-deoxy derivative 5. Hydroxyl and fluoro groups at C-1' show a tendency to enhance the intramolecular transglucosylations, giving 3-O-(alpha-D-glucopyranosyl)-D-fructose derivatives.

Bacteria↗