Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid

Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets (Solanumtuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12-³H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.     

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli/

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067–1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.     

Carminic acid, a non-competitive inhibitor of kidney UDP-glucose:galactosylhydroxylysine-collagen glucosyltransferase

1. UDP-glucose:galactosylhydroxylsine-collagen glucosyltransferase was purified 12-fold from rat kidney. 2. An assay using calf-skin gelatin as substrate showed time- and enzyme-dependent incorporation; KmS for UDP-glucose and gelatin were 16-7 microM and 4.5 mg/ml, respectively. 3. Column chromatography of the alkaline hydrolysate of reaction product on Dowex 50W-4X(H+) showed that 84% of the radioactivity was in the glycosylgalactosylhydroxylsine peak. 4. Carminic acid inhibited collagen glycosyltransferase; a dose-dependent study showed a two-stage inhibition and kinetic analysis by double-reciprocal plots at varying UDP-glucose concentrations revealed a non-competitive mode of inhibition.     

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.     

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters). Received: 14 July 2000 / Accepted: 8 August 2000     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Occurrence and characterization of a UDP-glucose:hydroxamic acid glucosyltransferase isolated from wheat (Triticum aestivum) seedlings

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat (Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2.4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N., and Iwamura H. (1995) Phytochemistry 38, 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.     

Isolation and characterization of UDP-glucose dolichyl-phosphate glucosyltransferase from human liver

The enzyme UDP-glucose dolichyl-phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100-fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion- and cation-exchange chromatography, 5-HgUDP-thiopropyl-Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl-phosphate-dependent glycosyltransferases catalyzing the formation of dolichyl diphospho-N-acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36,000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity.     

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.     

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.     

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.     

Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.

A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.     

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.     

Organization of mitochondrial UDP-glucose: Dolichylmonophosphate glucosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.     

An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase

The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP-glucose α-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP-glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin-glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.     

Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack

Salicylic acid (SA) is absolutely required for establishment of acquired resistance in non-infected tissues following localized challenge of other leaves with a necrotizing pathogen. Although not directly responsive to SA, or induced systemically following pathogen challenge, the expression of defence gene promoter fusions AoPR1—GUS and PAL-3—GUS after wounding or pathogen challenge could be enhanced by pre-treating tobacco plants hydroponically with SA, a phenomenon designated 'potentiation'. Potentiation of AoPR1—GUS wound-responsiveness was also demonstrated locally, but not systemically, in tobacco tissue exhibiting acquired resistance following infection with either viral or bacterial pathogens. Potentiation of wound-responsive expression by prior wounding could not be demonstrated. In contrast, potentiation of pathogen-responsive AoPR1—GUS expression was exhibited both locally and systemically in non-infected tissue. The spatial and temporal exhibition of defence gene potentiation correlated directly with the acquisition of resistance in non-infected tissue. Pathogen-responsive potentiation was obtained at about 10-fold lower levels of salicylic acid than wounding-responsive potentiation in AoPR1—GUS tobacco plants prefed with salicylate. These results may explain the failure to observe systemic potentiation of the wound-responsive defence gene expression. The data suggest a dual role for SA in terms of gene induction in acquired immunity: a direct one by induction of genes such as pathogenesis-related proteins, and an indirect one by potentiation of expression of other local defence genes (such as PAL and AoPR1) which do not respond directly to SA but become induced on pathogen attack or wounding.     

Purification, cloning, and expression of the prolactin receptor

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.