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PubMed1990

Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland.

UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.

Affinity Labels↗

PubMed1983

Partial purification and properties of a murine plasmacytoma glucosyltransferase.

The enzyme UDP-glucose:dolichylphosphate glucosyltransferase has been purified 1700-fold from MOPC-315 plasmacytoma tissue. The purification combines differential detergent extraction of purified rough endoplasmic reticulum with subsequent ion exchange chromatography, dye affinity chromatography, and hydroxylapatite chromatography of the extract. The partially purified glucosyltransferase exhibits a Km of 0.79 microM for UDP-Glc and a Km of 0.65 microM for dolicholphosphate in the presence of 4 mg/ml of phosphatidylcholine. The reaction is dependent upon the addition of exogenous dolicholphosphate. The enzyme is activated by the choline containing lipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The dye Remazol blue acts as a competitive inhibitor of the enzyme with respect to UDP-Glc. The molecular weight of the enzyme has been determined to be approximately 37,000. The sole reaction product has been identified as dolichylphosphate glucose by isolation of the product by DEAE-cellulose chromatography and subsequent analysis of the acid-hydrolyzed product by both Bio-Gel P2 gel filtration and paper chromatography.

Animals↗

PubMed1984

Biosynthesis of membrane-derived oligosaccharides. Novel glucosyltransferase system from Escherichia coli for the elongation of beta 1----2-linked polyglucose chains.

Membrane-derived oligosaccharides (MDO) of Escherichia coli are a family of substituted branched oligomers containing 8-12 residues of glucose that are joined by beta 1----2 and beta 1----6 linkages. MDO are localized in the periplasmic space of the cell, and their biosynthesis is regulated by the osmolarity of the medium (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095). We report here the initial characterization of a novel glucosyltransferase system that catalyzes the elongation of beta 1----2-linked polyglucose chains. The system requires: 1) a beta-D-glucoside such as the disaccharide sophorose (2-O-beta-D-glucosyl-glucose) or octyl beta-D-glucoside; 2) a trypsin-sensitive membrane fraction; 3) a heat-stable protein from the soluble fraction; 4) UDP-glucose; and 5) Mg2+ ions. Oligomers containing 6-10 glucose units (about the same size as MDO) that are joined by beta 1----2 linkages are major products of the enzyme system. Mutants in the recently mapped mdoA locus (Bohin, J. -P., and Kennedy, E. P. (1984) J. Bacteriol. 157, 956-957) are blocked in vivo at an early stage of MDO synthesis. It has now been found that mdoA mutants are defective in the membrane component, but not in the heat-stable protein that is required for the in vitro synthesis of beta 1----2-linked glucosyl oligomers. We conclude that the glucosyltransferase system described here has an essential function in the synthesis of MDO in vivo.

Cations, Divalent↗

PubMed1977

Effects of local immunization with glucosyltransferase fractions from Streptococcus mutans on dental caries in rats and hamsters.

The effect of local immunization with glucosyltransferase enzymes of Streptococcus mutans on dental caries in conventional rats, hamsters, and gnotobiotic rats was studied. Injection of these animals with crude or defined glucosyltransferase enzyme preparations incorporated into complete Freund's adjuvant consistently resulted in the presence of antibody in saliva demonstrable by functional inhibition of enzymatic activity and binding of radioactive enzyme. Serum antibody was also present. Two experiments have been completed in conventional rats and one in gnotobiotic rats in which the animals were immunized with crude enzyme antigens. The immunized groups of animals always had lower mean caries scores than comparably sham-immunized or nonimmunized control groups. Hamsters immunized with a defined enzyme preparation, containing no more than three antigenic components (two of which were enzyme), also demonstrated significant reductions in mean caries scores. The numbers of lesions were also always lower in immunized animals. In some cases there were reductions in the numbers of S. mutans that could be recovered from the teeth of immunized, infected animals. The reductions in dental caries and lesions were greater on smooth dental surfaces than on occlusal surfaces, which might be explained as interference with adherence phenomena demonstrated by S. mutans. It is proposed that antibody interference affects dental caries caused by this organism.

Animals↗

PubMed1997

UDP-glucose:solasodine glucosyltransferase from eggplant (Solanum melongena L.) leaves: partial purification and characterization.

Uridine 5'-diphosphoglucose-dependent glucosyltransferase which catalyzes the glycosylation of solasodine i.e. UDP-glucose:solasodine glucosyltransferase, is present in leaves, roots, unripe fruits and unripe seeds of eggplant (Solanum melongena L.). The glucosylation product is chromatographically identical with authentic solasodine 3 beta-D-monoglucoside, a putative intermediate in the biosynthesis of solasodine-based glycoalkaloids characteristic of the eggplant. The enzyme was purified about 50-fold from crude cytosol fraction of eggplant leaves by ammonium sulphate precipitation and column chromatography on Q-Sepharose and Sephadex G-100. The native enzyme has a molecular mass of approx. 55 kDa and pH optimum of 8.5. Divalent metal ions are not required for its activity but the presence of free-SH groups is essential. Besides solasodine (Km = 0.04 microM), the enzyme effectively glucosylates tomatidine, another steroidal alkaloid of the spirosolane type, but it is virtually inactive towards the solanidane-type steroidal alkaloids such as solanidine or demissidine. The enzyme is specific for UDP-glucose (Km = 2.1 microM) since unlabelled ADP-, GDP-, CDP- or TDP-glucose could not effectively compete with UDP-[14C]glucose used as the sugar donor for solasodine glucosylation. Moreover, no synthesis of labelled solasodine galactoside was observed when UDP-[14C]glucose was replaced with UDP-[14C]galactose.

Glucosyltransferases↗

PubMed1994

Lipoteichoic acid from Streptococcus sanguis is a natural glucosyl acceptor for glucosyltransferases.

The synthesis of methanol insoluble-[14C]labeled-polymers from [14C]sucrose by S. mutans glucosyltransferases was stimulated up to four-fold by the presence of highly purified, lipoteichoic acid (LTA) from S. sanguis. Gel filtration chromatography (Sepharose 4B) and ion exchange chromatography (DEAE-Bio-Gel A) of the [14C]labeled-polymers formed in the presence of [3H]glycerol-labeled-LTA showed that high molecular weight, negatively charged [14C]labeled-[3H]glycerol-labeled-LTA complexes were being formed. The [14C]component was identified as glucose by acid hydrolysis and paper chromatography. A similar high molecular weight, negatively charged [14C]glucosyl-[3H]glycerol-labeled-polymer was extracted from S. mutans cells grown in the presence of [3H]glycerol and [14C]sucrose, suggesting that LTA is a glucosyl-acceptor for glucosyltransferases in vivo as well as in vitro.

Chromatography, Gel↗

PubMed1996

A circularly permuted alpha-amylase-type alpha/beta-barrel structure in glucan-synthesizing glucosyltransferases.

A motif of amino acid residues, located at the active site and specific beta-strands in alpha-amylases, is recognized in alpha-1,3- and alpha-1,6-glucan-synthesizing glucosyltransferases, leading to the conclusion that these enzymes contain an alpha/beta-barrel closely related to the (beta/alpha)8-fold of the alpha-amylase superfamily. The secondary structure elements of the transferase barrel, however, are circularly permuted to start with an alpha-helix equivalent to helix 3 in the alpha-amylases. Thus, the transferase counterpart of the long third beta-->alpha connection--constituting a domain in the alpha-amylases--is divided to precede and succeed the barrel. This architectural arrangement may be coupled to sucrose scission and glucosyl transfer. The involvement in the mechanism in glucosyltransferases of active site residues recurring in amylolytic enzymes is discussed.

Amino Acid Sequence↗

PubMed2000

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells.

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn(2+), Co(2+) and Cu(2+) ions, as well as by SH-blocking reagents. The K(m) values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

Journal Article↗

PubMed2000

Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity.

UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality control by preventing transport of improperly folded glycoproteins out of the ER. We report the isolation of two cDNAs encoding human UDP-glucose:glycoprotein glucosyltransferase homologues. HUGT1 encodes a 1555 amino acid polypeptide that, upon cleavage of an N-terminal signal peptide, is predicted to produce a soluble 173 kDa protein with the ER retrieval signal REEL. HUGT2 encodes a 1516 amino acid polypeptide that also contains a signal peptide and the ER retrieval signal HDEL. HUGT1 shares 55% identity with HUGT2 and 31-45% identity with Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe homologues, with most extensive conservation of residues in the carboxy-terminal fifth of the protein, the proposed catalytic domain. HUGT1 is expressed as multiple mRNA species that are induced to similar extents upon disruption of protein folding in the ER. In contrast, HUGT2 is transcribed as a single mRNA species that is not induced under similar conditions. HUGT1 and HUGT2 mRNAs are broadly expressed in multiple tissues and differ slightly in their tissue distribution. The HUGT1 and HUGT2 cDNAs were expressed by transient transfection in COS-1 monkey cells to obtain similar levels of protein localized to the ER. Extracts from HUGT1-transfected cells displayed a 27-fold increase in the transfer of [(14)C]glucose from UDP-[(14)C]glucose to denatured substrates. Despite its high degree of sequence identity with HUGT1, the expressed recombinant HUGT2 protein was not functional under the conditions optimized for HUGT1. Site-directed alanine mutagenesis within a highly conserved region of HUGT1 identified four residues that are essential for catalytic function.

Amino Acid Sequence↗

PubMed2000

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase.

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.

Arabidopsis↗

PubMed2003

The noncatalytic portion of human UDP-glucose: glycoprotein glucosyltransferase I confers UDP-glucose binding and transferase function to the catalytic domain.

The eukaryotic cell monitors the fidelity of protein folding in the endoplasmic reticulum and only permits properly folded and/or assembled proteins to transit to the Golgi compartment in a process termed "quality control." An endoplasmic reticulum (ER) lumenal sensor for quality control is the UDP-glucose:glycoprotein glucosyltransferase that targets unfolded glycoproteins for transient, calcium-dependent glucosylation. This modification mediates glycoprotein interaction with the folding machinery comprised of calnexin or calreticulin in conjunction with ERp57. Two human UGT homologues, HUGT1 and HUGT2, exist that share 55% identity. The highest degree of identity resides in the COOH-terminal 20% of these proteins, the putative catalytic domain of HUGT1. However, only HUGT1 displays the expected functional activity. The contribution of the NH2-terminal remainder of HUGT1 to glucosyltransferase function is presently unknown. In this report we demonstrate that HUGT2 is localized to the ER in a manner that overlaps the distribution of HUGT1. Analysis of a series of HUGT1 and HUGT2 chimeric proteins demonstrated that the carboxyl-terminal region of HUGT2 contains a catalytic domain that is functional in place of the analogous portion of HUGT1. Whereas neither catalytic domain displayed detectable activity when expressed alone, co-expression of either catalytic domain with the noncatalytic amino-terminal portion of HUGT1 conferred UDP-Glc binding and transfer of glucose that was specific for unfolded glycoprotein substrates. The results indicate that the amino-terminal 80% of HUGT1 is required for activation of the catalytic domain, whereas the homologous portion of HUGT2 cannot provide this function.

Amino Acid Sequence↗

PubMed1992

Partial purification and properties of an inducible uridine 5'-diphosphate-glucose-salicylic Acid glucosyltransferase from oat roots.

A salicylic acid (SA)-inducible uridine 5'-diphosphate (UDP)-glucose:SA 3-O-glucosyltransferase was extracted from oat (Avena sativa L. cv Dal) roots. Reverse phase high-performance liquid chromatography or anion exchange chromatography was used to separate SA from the product, beta-O-d-glucosylsalicylic acid. The soluble enzyme was purified 176-fold with 5% recovery using a combination of pH fractionation, anion exchange, gel filtration, and chromatofocusing chromatography. The partially purified protein had a native molecular weight of about 50,000, an apparent isoelectric point at pH 5.0, and maximum activity at pH 5.5. The enzyme had a K(m) of 0.28 mm for UDP-glucose and was highly specific for this sugar donor. More than 20 hydroxybenzoic and hydroxycinnamic acid derivatives were assayed as potential glucose acceptors. UDP-glucose:SA 3-O-glucosyltransferase activity was highly specific toward SA (K(m) = 0.16 mm). The enzyme was inhibited by UDP and uridine 5'-triphosphate but not by up to 7.5 mm uridine 5'-monophosphate.

Journal Article↗

PubMed1989

Hydroxamic Acid glucosyltransferases from maize seedlings.

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K(m) of 174 micromolar for DIMBOA and a K(m) of 638 micromolar for DIBOA; the enzyme in peak 1 has a K(m) of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K(m). The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.

Journal Article↗

PubMed1991

Isolation and Characterization of a UDPGlucose: Flavonol O-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings.

A UDPGlc:flavonol O(3)-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol O(3)-glucosides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH(4))(2)SO(4)-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH optimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M(r) of 59,000, and it was composed of two similar subunits of M(r) 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (K(m)~12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis.

Journal Article↗

PubMed2000

Effects of resin composite components on glucosyltransferase of cariogenic bacterium.

The aim of our study was to investigate the effect of resin composite components on glucan synthesis by glucosyltransferase (GTase) derived from a cariogenic bacterium, Streptococcus sobrinus B13. The eluates from cured composites stored in 0.05 mol/L potassium phosphate buffer (KPB) (pH 6.8) for 2 weeks at 37 degrees C stimulated the formation of water-insoluble glucan, whereas those from amalgam inhibited it. This finding suggests that the eluates from the resin composites enhance GTase activity and contribute to plaque formation. In the individual resin components, a diglycidyl methacrylate and triethylene glycol dimethacrylate significantly enhanced GTase activity. However, most polymerization inhibitors and accelerators were potent inhibitors of enzyme activity. Thus, GTase stimulation by eluates from resin composites is hypothesized to be heavily dependent on the effect of the eluated resin monomers, even though the other eluting components had inhibitory effects on GTase.

Composite Resins↗

PubMed2000

Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering of reaction conditions.

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1, 2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.

Biotechnology↗

PubMed2005

Development of a rapid, quantitative glucosyltransferase assay based on a screen-printed fructose enzyme electrode and application to optimization studies on gtfD expression in recombinant Escherichia coli.

A biosensor for fructose determination was used as basis of an assay for the determination of glucosyltransferase (GTF) activities and applied to monitoring recombinant enzyme production. GTFs catalyze the synthesis of glucans from sucrose leading to the release of fructose. Specific fructose determinations in the microM concentration range were achieved with a fructose electrode based on fructose dehydrogenase, which was immobilized on a screen-printed platinum electrode. This electrode was used as basis of the new assay for GTF activity determinations. Depending on the amount of enzyme, the assay was completed within 15-30 min compared to 1-2 h for the traditional photometric assay. From the amount of fructose released in a given reaction time, GTF activities were determined down to approx. 20 U/L. Even unpurified samples from a recombinant GTF-S production process could be analyzed without any problems, and a good correlation was obtained to data obtained from the photometric assay. Analysis of samples from cultures of various rGTF-S-producing recombinant E. coli strains grown on different media with SDS-PAGE and with the new assay identified the same strain and culture medium as optimum for recombinant GTF-S production.

Biosensing Techniques↗

PubMed2004

Identification of an UDP-Glc:glycoprotein glucosyltransferase in the yeast Yarrowia lipolytica.

The UDP-Glc:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that plays a determining part in the mechanism by which unfolded, partially folded or misfolded glycoproteins are retained into the ER. We have identified an UGT in the yeast Yarrowia lipolytica. This protein, of a predicted molecular weight of 165.7 kDa, is encoded by a 5054 bp coding sequence containing a 643 bp intron at position 682-1323. The N-terminal part of the protein displays a signal sequence whereas its C-terminal part carries an ER retrieval signal HDEL. An interruption of the gene that removes the 1075 last nucleotides of its sequence did not lead to any evident phenotype except for a slight increased sensitivity to tunicamycin. YlUGT1 mRNA levels respond to tunicamycin treatment by an induction factor of 2-4, which indicates that the gene product participates in the quality control mechanism in this yeast. Finally, an immunofluorescence study of the protein localization, shows that the protein distribution is different from that of previously studied ER resident proteins. This could indicate that UGT distribution in the secretory pathway is not confined to the ER.

Amino Acid Sequence↗