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PubMed1993

Purification and properties of UDP-glucose:thiohydroximate glucosyltransferase from Brassica napus L. seedlings.

A uridinediphosphateglucose:thiohydroximate glucosyltransferase (EC 2.4.1.-) has been purified 3700-fold from Brassica napus L. seedlings. The enzyme catalyzes the formation of desulfoglucosinolates by transfer of glucose from UDP-glucose to thiohydroximates and is believed to be the second to last step involved in glucosinolate biosynthesis. The enzyme was purified to near homogeneity, exhibiting a single band by non-denaturing polyacrylamide gel electrophoresis (PAGE) and on sodium dodecyl sulfate-PAGE (M(r) 46,000) but showed multiple isoforms between pH 4.6 and 4.3 when resolved by IEF. The enzyme is stable at temperatures up to 30 degrees C for at least 1 h and shows maximum activity rates at pH 6.0 and has no absolute requirements for cations. The Km values for UDP-glucose and phenylacetothiohydroximate were calculated to be 0.46 and 0.05 mM, respectively. This enzyme possesses a high degree of specificity for the thiohydroximic functional group but little specificity for the associated side-chain groups. Similar enzyme activity has been detected in all other members of the Brassicaceae family tested and is believed to be a common thiohydroximate glucosylating enzyme present in these and other glucosinolate producing plants.

Brassica↗

PubMed1995

Steviol and steviol-glycoside: glucosyltransferase activities in Stevia rebaudiana Bertoni--purification and partial characterization.

The leaves of Stevia rebaudiana Bertoni contain sweet compounds which are glycosides of diterpene derivative steviol (ent-13-hydroxykaur-16-en-19-oic acid). Its main constituents are stevioside (triglucosylated steviol; 13-O-beta-sophorosyl-19-O-beta-glucosyl-steviol) and rebaudioside-A (tetraglucosylated steviol; 2'-O-beta-glucosyl-13-O-beta-sophorosyl-19-O-beta-glucosyl-stev iol). From the extracts of S. rebaudiana Bertoni, two glucosyltransferases (GTases I and IIB) acting on steviol and steviol-glycosides were isolated, and another distinct activity (GTase IIA) acting on steviol was detected. Purified GTase I (subunit M(r) 24,600) catalyzed glucose transfer from UDP-glucose to steviol and steviolmonoside (steviol-13-O-glucopyranoside), but not to other steviol-glycosides. Apparent Km values were 71.4 microM for steviol and 360 microM for UDP-glucose. GTase IIB (subunit M(r) 30,700) showed a broad substrate specificity, acting on steviol, steviolmonoside, steviolbioside (13-O-beta-sophorosyl-steviol), and stevioside. Apparent Km values were 182 microM for steviol, 44 microM for steviolbioside, 95 microM for stevioside, and 385 microM for UDP-glucose. The two enzymes had a similar optimum pH at 6.5. They also acted effectively on ubiquitous flavonol aglycones, quercetin, and kaempferol and utilized kaempferol at a higher rate than steviol and steviol-glycosides. The apparent Km values of GTase I and IIB for kaempferol were 12 and 31 microM, respectively.

Chromatography, Gel↗

PubMed2001

A stress-responsive gene from groundnut, Gdi-15, is homologous to flavonol 3-O-glucosyltransferase involved in anthocyanin biosynthesis.

Stress-tolerant crops are expected to express genes not normally expressed in susceptible crops. We have used desiccation stress coupled with high light intensity to identify groundnut as a relatively tolerant crop. Stress-responsive genes (Gdi, Groundnut Desiccation Induced) were cloned by subtractive hybridisation. The sequence of Gdi-15 shows homology to flavonol 3-O-glucosyltransferases, which are involved in anthocyanin biosynthesis. Gdi-15 transcripts increase markedly in response to stress, suggestive of a role in stress tolerance.

Amino Acid Sequence↗

PubMed1999

T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 which catalyses the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection system aimed at host nucleases. We previously reported the first three-dimensional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regions (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate UDP-Glc (form II), respectively. Form I crystals are grown in ammonium sulphate and the structure has been determined to 1.88 A resolution (R -factor 19.1 %). Form II crystals are grown in polyethyleneglycol 4000 and the structure has been solved to 2.3 A resolution (R -factor 19.8 %). The form I structure is isomorphous to our previous BGT UDP-Glc structure. The form II structure, however, has allowed us to model the two missing surface loop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data however, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, which probably accounts for the absence of Glc in our UDP-Glc substrate structures. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity between the catalytic core of glycogen phosphorylase (GP) and BGT, we have modelled the position of the Glc moiety in UDP-Glc. From these two models, we propose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide.

Bacteriophage T4↗

PubMed2001

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.

Allosteric Site↗

PubMed1996

Deletion and reintroduction of glucosyltransferase genes of Streptococcus mutans and role of their gene products in sucrose dependent cellular adherence.

Streptococcus mutans has three kinds of glucosyltransferases (GTases), i.e. GTase-I, GTase-SI and GTase-S. These enzymes co-operatively synthesize adhesive glucan from sucrose, which contributes to firm adherence of growing organisms to solid surfaces. In this study, the genes encoding GTase-I (gtfB) and GTase-SI (gtfC) of S. mutans MT8148 (serotype c) were inactivated by the insertion mutagenesis by allelic exchange. Three types of isogenic mutants lacking either GTase-I, GTase-SI or both, respectively were isolated. Sucrose dependent cellular adherence of these mutants were significantly lower than that of the parent. Adherence of GTase-SI deficient mutant was lower than that of GTase-I deficient mutant. We then generated Escherichia coli-Streptococcus shuttle vectors carrying the gtfB and gtfC gene. The shuttle vector containing the gtfB gene was transformed into the GTase-I deficient mutant. Western blot analysis of the transformant revealed that GTase-I protein was fully expressed. Sucrose dependent adherence of the transformant increased but did not reach that of the parent. Similarly, the shuttle vector containing the gtfC gene was transformed into the GTase-SI deficient mutant. The expression of GTase-SI and sucrose dependent adherence of the transformant was revealed to be at a level similar to those by the parent. These results indicated that GTase-SI does play an essential role in the production of adhesive glucan that can lead to firm cellular adherence to solid surfaces.

Bacterial Adhesion↗

PubMed1978

Cross-protective aspects of glucosyltransferase antigens in the hamster caries model.

1) Glucosyltransferase serotypes a and g are closely related antigenically but are more distantly related to GTF of serotype c, based on assays of inhibition of total glucan synthesis. 2) Local immunization with GTF from serotype c S. mutans reduces the colonization, caries, and lesions caused by infection with the homologous strain compared with sham injected controls. 3) Local immunization with GTF of serotype c S. mutans reduces the colonization, caries, and lesions caused by infection with S. mutans of serotype g (strain 6715).

Animals↗

PubMed1987

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans.

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.

Electrophoresis, Polyacrylamide Gel↗

PubMed1984

Influence of B and Pl on UDPG:Flavonoid-3-O-glucosyltransferase in Zea mays L.

The influence of the genetic constitution at the B and Pl loci on UDPG:flavonoid-3-O-glucosyltransferase activity is described. More than a 90% reduction in activity is found when either B or Pl was present in the homozygous recessive condition. A positive correlation between quercetin-3-O-glucoside and pelargonidin-3-O-glucoside production is observed for all genotypes tested. Changes in UFGT activity during plant development are described for R-r B Pl plants.

Anthocyanins↗

PubMed1977

Genetic control of UDPglucose:flavonol 3-O-glucosyltransferase in the endosperm of maize.

The enzyme UDPglucose:flavonol 3-O-glucosyltransferase is shown to be under the coordinate control of three genes involved in anthocyanin biosynthesis in the aleurone of maize: C, R, and BZ. Of the three, BZ appears to be the structural gene. Data presented here (dosage comparisons, induction in the mutant c-p, and effect of paramutation at R) indicate that the enzyme is inducible by substances resulting from the action of the C and R genes and that active forms of C and R are required for this induction. Mechanisms of regulation of the BZ gene by C and R are discussed.

Flavonoids↗

PubMed1983

Increased activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, enzymes of collagen biosynthesis, in skeletal muscle of endurance-trained mice.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), and the concentration of 4-hydroxyproline were measured in red and white parts of quadriceps femoris muscle of mice after 3, 10, and 20 sessions of daily endurance training. The activities of PH and GGT increased in the red part of the muscle after training for 3 and 10 times and returned to the control level after 20 training sessions. In the white muscle the increase of PH activity was less than in the red muscle. No alteration in GGT activity was observed in the white muscle. The concentration of hydroxyproline was unchanged in the both types of skeletal muscle. The results suggest that collagen turnover in leg muscles may be enhanced during the early phase of adaptation to endurance training. The enhancement is more prominent in red than in white skeletal muscle.

Animals↗

PubMed1986

Prolonged exercise causes an increase in the activity of galactosylhydroxylysyl glucosyltransferase and in the concentration of type III procollagen aminopropeptide in human serum.

The effect of prolonged heavy physical exercise on serum galactosylhydroxylysyl glucosyltransferase activity (S-GGT) and serum type III procollagen aminoterminal propeptide (S-Pro(III)-N-P) concentration was studied in healthy male long-distance runners. S-GGT increased gradually by about 70% (p less than 0.01) during a competitive 24-h run, and a rising trend was also observed in S-Pro(III)-N-P. After the termination of the run S-GGT was normalized in two days, but the increase in S-Pro(III)-N-P continued up to one day after the race, reaching nearly 40% (p less than 0.005). The alterations in S-GGT and S-Pro(III)-N-P showed no significant correlation with S-CK, S-LDH or the distance run. The most likely explanation for the increases in S-GGT and S-Pro(III)-N-P is that prolonged heavy exercise injures the collagen-synthesizing cells of the connective tissue, leading to a short-term increase in type III procollagen production.

Adult↗

PubMed1994

Purification and properties of glucosyltransferase from Aureobasidium.

Purification and properties of glucosyltransferase, which produces panose (Glc alpha 1-->6Glc alpha 1-->4Glc) and isomaltose (Glc alpha 1-->6Glc) from maltose (Glc alpha 1-->4Glc), are reported. The enzyme, from Aureobasidium, was purified to homogeneity by fractionations involving ammonium sulfate and DEAE-Cellulofine, S-Sepharose Fast Flow and Sephadex G-200 chromatography. Molecular mass of the enzyme was estimated to be 395 kDa by gel filtration. The enzyme was identified as a glycoprotein which contains 32% (w/w) carbohydrate. The optimum pH for the enzymatic reaction was 4.5-5.5 and the enzyme was stable over a pH range of 4-6. The optimum reaction temperature for the enzyme was 65 degrees C and the enzyme retained more than 96% activity at 60 degrees C after 15 min. The enzyme produced panose from maltose by means of a high efficiency (45.5%) glucosyl-transfer reaction. The enzyme was inhibited by metal ions, such as those of mercury, silver and aluminum, and also by organic inhibitors, especially nitrilotriacetic acid.

Biotechnology↗

PubMed1995

Identification and nucleotide sequence of an ecdysteroid UDP-glucosyltransferase gene of Spodoptera littoralis multicapsid nuclear polyhedrosis virus.

The Spodoptera littoralis multicapsid nuclear polyhedrosis virus (SlMNPV) is a member of the Baculoviridae that shows a distant genetic relationship to the prototype Autographa californica MNPV (AcMNPV). Using an AcMNPV gene-specific probe, we identified and mapped an ecdysteroid UDP-glucosyltransferase (egt) gene in the genome of SlMNPV. Sequence determination of a part from the hybridizing DNA fragment revealed an open reading frame of 1548 nucleotides that exhibits 38% and 44% identity to the egt amino acid sequences of AcMNPV and Lymantria dispar MNPV (LdMNPV), respectively. Sequences flanking the SlMNPV egt gene, including the promoter region, were found to be unique to the virus. The presence of this nonstructural gene in SlMNPV and several other baculoviruses points to the importance of egt for the viral infection process.

Amino Acid Sequence↗

PubMed1976

Elevated kidney glucosyltransferase activity in genetic prediabetic mice.

Glucosyltransferase activity in the renal cortex of genetic diabetic KK mice was significantly increased at 40 days of age when compared to that of Swiss albino and F1 hybrid mice. This increase in enzyme activity in the absence of glucose intolerance can be regarded as an earlier genetic marker for the diagnosis of diabetic microangiopathy.

Age Factors↗

PubMed1979

Partial purification and properties of glucosyltransferase from Streptomyces aureofaciens.

Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase from Streptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 microM for 1,2-dihydroxy-9,10-anthraquinone (alizarin) 110 microM; the maximum rate of glucosylation reaction was 5.32 mumol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 degrees C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with alpha-hydroxyl groups was about 1/8 of the values obtained with beta-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.

Anthraquinones↗

PubMed2004

Isolation and characterization of a novel intracellular glucosyltransferase from the acarbose producer Actinoplanes sp. CKD485-16.

A novel intracellular glucosyltransferase (GTase) was isolated from cells of Actinoplanes sp. CKD485-16-acarbose-producing cells. The enzyme was purified by DEAE-cellulose and G75-40 Sephadex chromatography. The molecular mass of the enzyme was estimated to be 62 kDa by SDS-polyacrylamide gel electrophoresis, and its isoelectric point (pI) was pH 4.3. The N-terminal sequence of the GTase consisted of NH(2)-Ser-Val-Pro-Leu-Ser-Leu-Pro-Ala-Glu-Trp. The optimum pH and temperature were 7.5 and 30 degrees C. The enzyme was stable in a pH range of 5.5-9.0 and below 40 degrees C. Enzymatic reactions were performed by incubating the GTase with various substrates. The GTase converted acarbose into component C, maltose into trehalose, and maltooligosaccharides into maltooligosyl trehaloses. The reactions were reversible. Various acarbose analogs were tested as inhibitors against the GTase as a means to suppress component C formation. Valienamine was the most potent, with an IC(50) value of 2.4x10(-3) mM and showed a competitive inhibition mode.

Acarbose↗

PubMed2003

Arabidopsis glucosyltransferases with activities toward both endogenous and xenobiotic substrates.

Arabidopsis thaliana Heynh. harbors UDP-glucose-dependent glucosyltransferase (UGT; EC 2.4.1.-) activities that are able to glucosylate xenobiotic substrates as a crucial step in their detoxification, similar to other plants. However, it has remained elusive whether side-activities of UGTs acting on endogenous substrates could account for that property. Therefore, seven recombinantly expressed A. thaliana enzymes were tested using the phytotoxic xenobiotic model compound 2,4,5-trichlorophenol (TCP) as a substrate. The enzymes were selected from the large Arabidopsis UGT gene family because their previously identified putative endogenous substrates comprised both carboxylic acid, and phenolic and aliphatic hydroxyl moieties as biochemical targets. In addition, UGT75D1, which was shown to accept the endogenous flavonoid kaempferol as a substrate, was included. All enzymes tested, except the sterol-conjugating UGT80A2, glucosylated TCP as a parallel activity. The K(m) values for TCP ranged from 0.059 to 1.25 mM. When tested at saturating concentrations of the native substrates the glucosylation of TCP by the glucose-ester-forming UGT84A1 and UGT84A2 was suppressed by p-coumaric acid and sinapic acid, respectively. In contrast, the activities of UGT72E2 and UGT75D1 toward their phenolic native substrates and the xenobiotic TCP were mutually inhibited. TCP was a competitive inhibitor of sinapyl alcohol glucosylation by UGT72E2. These overlapping in vitro activities suggest cross-talk between the detoxification of xenobiotics and endogenous metabolism at the biochemical level, depending on the presence of competing substrates and enzymes.

Arabidopsis↗