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PubMed1991

Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975.

The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage lambda L47.1. The inserts in the lambda-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.

Amino Acid Sequence↗

PubMed1991

Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase.

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2.6-fold and 4.7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.

Electrophoresis, Polyacrylamide Gel↗

PubMed1991

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Bacterial Proteins↗

PubMed2002

Role of the C-terminal YG repeats of the primer-dependent streptococcal glucosyltransferase, GtfJ, in binding to dextran and mutan.

The recombinant primer-dependent glucosyltransferase GtfJ of Streptococcus salivarius possesses a C-terminal glucan-binding domain composed of eighteen 21 aa YG repeats. By engineering a series of C-terminal truncated proteins, the position at which truncation prevented further mutan synthesis was defined to a region of 43 aa, confirming that not all of the YG motifs were required for the formation of mutan by GtfJ. The role of the YG repeats in glucan binding was investigated in detail. Three proteins consisting of 3.8, 7.2 or 11.0 C-terminal YG repeats were expressed in Escherichia coli. Each of the three purified proteins bound to both the 1,6-alpha-linked glucose residues of dextran and the 1,3-alpha-linked glucose residues of mutan, indicating that a protein consisting of nothing but 3.8 YG repeats could attach to either substrate. Secondary structure predictions of the primary amino acid sequence suggested that 37% of the amino acids were capable of forming a structure such that five regions of beta-sheet were separated by regions capable of forming beta-turns and random coils. CD spectral analysis showed that the purified 3.8 YG protein possessed an unordered secondary structure with some evidence of possible beta-sheet formation and that the protein maintained this relatively unordered structure on binding to dextran.

Amino Acid Motifs↗

PubMed1993

Effects of selected surfactants on purified glucosyltransferases from mutans streptococci and cellular adherence to smooth surfaces.

The inhibitory effect of non-ionic, anionic, cationic and ampholytic surfactants on cellular growth of Streptococcus mutans MT8148 and S. sobrinus 6715, on glucan synthesis by the purified glucosyltransferase (GTase) from these organisms, and on bacterial adherence to glass surfaces was examined in vitro. Cationic surfactants exhibited marked bactericidal activities. Anionic and ampholytic compounds were less strongly bactericidal and non-ionic surfactants produced only slight inhibition of cell growth under the conditions tested. Some non-ionic compounds had no effect on this. Glucan synthesis by GTase from mutans streptococci was inhibited by anionic and cationic surfactants. Among various GTase proteins, insoluble glucan synthesising GTases, i.e., S. mutans CA-GTase and S. sobrinus GTase-I were those most effectively inhibited by these agents. However, it was noted that whereas lower concentrations of cationic surfactants enhanced these GTase activities, higher concentrations of the surfactants were inhibitory. Non-ionic detergents stimulated soluble glucan synthesis from S. mutans CF-GTase and cationic and ampholytic surfactants enhanced or inhibited glucan synthesis depending on the concentrations of the surfactants. Sucrose-dependent cellular adherence of resting cells of mutans streptococci to glass surfaces was inhibited by the addition of surfactants that annulled the GTase activities.

Bacterial Adhesion↗

PubMed1995

Four glucosyltransferases, GtfJ, GtfK, GtfL and GtfM, from Streptococcus salivarius ATCC 25975.

The four recombinant glucosyltransferases (GTFs), GtfJ, GtfK, GtfL and GtfM, that had previously been cloned from Streptococcus salivarius ATCC 25975, were individually expressed in Escherichia coli and their glucan products and kinetic properties were analysed. GtfJ was a primer-dependent GTF which synthesized an insoluble glucan composed mainly of alpha-(1-->3)-linked glucosyl residues in the presence of dextran T-10. GtfK was primer-stimulated, and produced a linear soluble dextran without any detectable branch points both in the absence and in the presence of dextran T-10. GtfL was primer-independent and produced a mixed-linkage insoluble glucan composed of approximately equal proportions of alpha-(1-->3)- and alpha-(1-->6)-linked glucosyl residues. GtfL was inhibited by dextran T-10. GtfM was primer-independent and produced a soluble dextran with approximately 5% alpha-(1-->3)-linked glucosyl residues. GtfM was essentially unaffected by the presence of dextran T-10. The results confirmed that each enzyme represented one of the four possible combinations of primer-dependency and product solubility and that each possessed unique biosynthetic properties. The soluble dextrans formed by GtfK and GtfM, as well as the mixed-linkage insoluble glucan formed by GtfL, were also capable of acting as primers for the primer-dependent GtfJ and the primer-stimulated GtfK. Unexpectedly, the linear dextran produced by GtfK was by far the least effective either at priming itself or at activating and priming the primer-dependent GtfJ.

Bacterial Proteins↗

PubMed2007

Structural and genetic evidence that the Escherichia coli O148 O antigen is the precursor of the Shigella dysenteriae type 1 O antigen and identification of a glucosyltransferase gene.

Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpNAc-(1-->. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (Glc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 1 O antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.

Bacterial Proteins↗

PubMed2002

Cloning and characterization of the abscisic acid-specific glucosyltransferase gene from adzuki bean seedlings.

The glycosylated forms of abscisic acid (ABA) have been identified from many plant species and are known to be the forms of ABA-catabolism, although their (physiological) roles have not yet been elucidated. ABA-glucosyltransferase (-GTase) is thought to play a key role in the glycosylation of ABA. We isolated an ABA-inducible GTase gene from UDP-GTase homologs obtained from adzuki bean (Vigna angularis) seedlings. The deduced amino acid sequence (accession no. AB065190) showed 30% to 44% identity with the known UDP-GTase homologs. The recombinant protein with a glutathione S-transferase-tag was expressed in Escherichia coli and showed enzymatic activity in an ABA-specific manner. The enzymatic activity was detected over a wide pH range from 5.0 to 9.0, the optimum range being between pH 6.0 and 7.3, in a citrate and Tris-HCl buffer. The product from racemic ABA and UDP-D-glucose was identified to be ABA-GE by gas chromatography/mass spectrometry. The recombinant GTase (rAOG) converted 2-trans-(+)-ABA better than (+)-S-ABA and (-)-R-ABA. Although trans-cinnamic acid was slightly converted to its conjugate by the GTase, (-)-PA was not at all. The mRNA level was increased by ABA application or by water stress and wounding. We suggest that the gene encodes an ABA-specific GTase and that its expression is regulated by environmental stress.

Abscisic Acid↗

PubMed2006

Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding beta-amyrin synthase and a triterpene carboxylic acid glucosyltransferase.

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a beta-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria.

Amino Acid Sequence↗

PubMed2002

Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress, and weakens virus resistance.

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.

Chlorogenic Acid↗

PubMed2002

Crystallization and preliminary crystallographic study of a ternary complex between the T4 phage beta-glucosyltransferase, uridine diphosphoglucose and a DNA fragment containing an abasic site.

A base-flipping phenomenon has been established for DNA methyltransferases and for DNA base-excision repair glycosylases and is likely to prove general for enzymes that need access to DNA bases to undergo chemical reaction. T4 phage beta-glucosyltransferase (BGT) is a good candidate for this novel mechanism. In order to confirm this, BGT was crystallized with an abasic site-containing DNA and uridine diphosphoglucose (UDP-glucose). The crystallization strategy is described. A complete data set was collected at 1.8 A resolution on a Cu Kalpha rotating-anode X-ray source. Molecular replacement was performed and the initial electron-density maps clearly show bound DNA.

Bacteriophage T4↗

PubMed1979

Serotype-dependent inhibition of glucan synthesis and cell adherence of Streptococcus mutans by antibody against glucosyltransferase of serotype e S. mutans.

A crude glucosyltransferase (GTase) preparation was obtained from the culture supernatant of Streptococcus mutans strain MT703 (serotype e) by 50% ammonium sulphate precipitation. Antiserum specific against the GTase was prepared by immunizing rabbits intramuscularly with the GTase in Freund incomplete adjuvant, followed by GTase without adjuvant intravenously. Gamma globulin fractions of the antiserum and normal serum were partially purified by 1/3 saturated ammonium sulphate precipitation. The antibody strongly inhibited the GTase activity (greater than 90%) of type c, e and f S. mutans, whereas the GTase of type a, d and g was not affected by the antibody. The GTase from type b S. mutans was slightly inhibited. The adherence of viable cells of type c, e, and f S. mutans to a glass surface due to synthesis of glucan by the cell-associated GTase was also significantly inhibited by the antibody to the enzyme. These results suggest that type c, e, and f and types a, d, and g S. mutans can be separated into two major groups in terms of the immunological relationship of GTase.

Antibodies, Bacterial↗

PubMed1983

Effect of maltose on glucan synthesis by glucosyltransferases of Streptococcus mutans.

The effects of added maltose on the activities of a preparation of crude glucosyltransferases (GTases) and purified dextransucrase (DS) were investigated to elucidate the inhibition mechanism of maltose on the synthesis of water-insoluble glucan (ISG) in Streptococcus mutans HS-6. Tri- and tetra-saccharides produced by crude GTases from sucrose in the presence of maltose were identified as panose (4-alpha-isomaltosylglucose) and 4-alpha-isomaltotriosylglucose which were responsible for the activity of DS involved in crude GTases. Kinetic studies on crude GTases in the presence of maltose showed similar results to those of DS except that the synthesis of ISG in the crude GTases was inhibited. Comparative studies of soluble products of crude GTases and DS in the presence of maltose were performed employing gel filtration on Sephadex G-15. The existence of oligosaccharides above hexasaccharide was revealed as the products of DS but not of crude GTases. These findings were interpreted in terms of the previously proposed mechanism of ISG synthesis by S. mutans, i.e., ISG should be synthesized from the preformed soluble glucan. It was indicated that oligosaccharides above hexasaccharide are utilized for ISG synthesis in the crude GTases system. From these results, the inhibitory mechanism of added maltose on ISG synthesis by crude GTases is considered as follows: DS synthesizes a series of 4-alpha-isomaltodextrinylglucose from sucrose and maltose, and the increase of added maltose results in the decrease of oligosaccharides responsible for synthesis of ISG.

Glucans↗

PubMed1982

Types I and III collagens and the activities of prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase in skin lesions of tuberous sclerosis.

Collagen synthesis and the ratios of collagen types I and III were assayed from the skin lesions of five subjects with tuberous sclerosis. Collagen synthesis, measured by the activities of prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase, was clearly increased in three angiofibromas of these patients and in one soft tumour of the face, but it was unchanged in shagreen patches. The total collagen content was decreased in angiofibromas, indicating either increased turnover of collagen or an increased amount of cellular or other macromolecular elements in these lesions. The proportions of types I and III collagens, estimated by cyanogenbromide digestion and SDS-gel electrophoresis, were 80-90% and 10-20%, respectively, in all samples except two angiofibromas, in which the relative amount of type III collagen was increased. This may indicate that angiofibromas of tuberous sclerosis are heterogenous with respect to the collagen types they contain, and that there may be disturbed cell growth or collagen synthesis, with individual variation from case to case.

Adult↗

PubMed1983

A local potent glucocorticosteroid decreases the induction of galactosylhydroxylysyl glucosyltransferase in suction blisters but has no effect on basement membrane structures.

The effects of local glucocorticosteroid treatment on collagen biosynthesis and basement membrane components were studied in suction blisters in human abdominal skin. Pretreatment with clobetasol-17-propionate, applied three times a day for 4 days, did not affect the activity of galactosylhydroxylysyl glucosyltransferase (GGT) in fresh blisters but post-blistering treatment for 3 days with the steroid markedly inhibited the increase of this enzyme activity during the initial phases of re-epithelialization. The GGT activity was over 50% lower in steroid-treated blisters compared with control values. Protein concentrations and blister fluid volumes were also significantly decreased in healing 3-day blisters after steroid treatment. These results suggest that local glucocorticosteroid decreases either the synthesis of GGT or its release from the tissue into the blister fluid. The treatment did not affect the blister histology nor the early process of re-epithelialization. Immunohistochemically, type IV and V collagens and laminin of the basement membrane zone were similarly located in blisters of steroid-treated and placebo-treated skin, suggesting that local glucocorticosteroid does not affect the integrity of the basement membrane.

Adult↗

PubMed2004

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night.

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.

Arabidopsis↗

PubMed2004

Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis.

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP-glucose:thiohydroximate S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low Km values for phenylacetothiohydroximic acid (approximately 6 microm) and UDP-glucose (approximately 50 microm) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss-of-function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light-grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole-3-acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.

Arabidopsis↗

PubMed2005

Functional importance of the family 1 glucosyltransferase UGT72B1 in the metabolism of xenobiotics in Arabidopsis thaliana.

The Arabidopsis type 1 UDP-glucose-dependent glucosyltransferase UGT72B1 is highly active in conjugating the persistent pollutants 3,4-dichloroaniline (DCA) and 2,4,5-trichlorophenol (TCP). To determine its importance in detoxifying xenobiotics in planta, mutant plants where the respective gene has been disrupted by T-DNA insertion have been characterized. Extracts from the knockout ugt72B1 plants showed radically reduced conjugating activity towards DCA and TCP and the absence of immunodetectable UGT72B1 protein. In contrast, activities towards phenolic natural products were unaffected. When aseptic root cultures were fed [14C]-DCA, compared with wild types, the ugt72B1 plants showed a reduced rate of uptake of the xenobiotic and very little metabolism to soluble DCA-glucose or associated polar conjugates. Instead, the knockouts accumulated non-extractable radioactive residues, most probably associated with lignification. When the feeding studies were carried out with [14C]-TCP, rates and routes of metabolism were identical in the wild type and knockouts, with TCP-glucoside a major product in both cases. Similar differential effects on the metabolism of DCA and TCP were obtained in whole plant studies with wild type and ugt72B1 mutants, demonstrating that while UGT72B1 had a central role in metabolizing chloroanilines in Arabidopsis, additional UGTs could compensate for the conjugation of TCP in the knockout. TCP was equally toxic to wild type and ugt72B1 plants, while surprisingly, the knockouts were less sensitive to DCA. From this it was concluded that the glucosylation of DCA may not be as effective in xenobiotic detoxification as bound-residue formation.

Aniline Compounds↗