Publications

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

  Reed D.W., Davin L., Jain J.C., Deluca V., Nelson L., Underhill E.W.

  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 las ... >> More

  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. << Less

  Arch Biochem Biophys 305:526-532(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A radioassay of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica species.

  Jain J.C., Reed D.W., GrootWassink J.W., Underhill E.W.

  A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate ... >> More

  A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate from anionic [14C]UDPglucose and anionic [14C]benzylglucosinolate, respectively, by differential adsorption to DEAE-ion-exchange disks. The procedure eliminates elaborate chromatographic techniques. The method was used to measure both enzymes in several Brassica spp. In addition, sulfotransferase activity was monitored during partial purification from seedlings of Brassica napus (cv Westar). << Less

  Anal Biochem 178:137-140(1989) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The chemical diversity and distribution of glucosinolates and isothiocyanates among plants.

  Fahey J.W., Zalcmann A.T., Talalay P.

  Glucosinolates (beta-thioglucoside-N-hydroxysulfates), the precursors of isothiocyanates, are present in sixteen families of dicotyledonous angiosperms including a large number of edible species. At least 120 different glucosinolates have been identified in these plants, although closely related t ... >> More

  Glucosinolates (beta-thioglucoside-N-hydroxysulfates), the precursors of isothiocyanates, are present in sixteen families of dicotyledonous angiosperms including a large number of edible species. At least 120 different glucosinolates have been identified in these plants, although closely related taxonomic groups typically contain only a small number of such compounds. Glucosinolates and/or their breakdown products have long been known for their fungicidal, bacteriocidal, nematocidal and allelopathic properties and have recently attracted intense research interest because of their cancer chemoprotective attributes. Numerous reviews have addressed the occurrence of glucosinolates in vegetables, primarily the family Brassicaceae (syn. Cruciferae; including Brassica spp and Raphanus spp). The major focus of much previous research has been on the negative aspects of these compounds because of the prevalence of certain "antinutritional" or goitrogenic glucosinolates in the protein-rich defatted meal from widely grown oilseed crops and in some domesticated vegetable crops. There is, however, an opposite and positive side of this picture represented by the therapeutic and prophylactic properties of other "nutritional" or "functional" glucosinolates. This review addresses the complex array of these biologically active and chemically diverse compounds many of which have been identified during the past three decades in other families. In addition to the Brassica vegetables, these glucosinolates have been found in hundreds of species, many of which are edible or could provide substantial quantities of glucosinolates for isolation, for biological evaluation, and potential application as chemoprotective or other dietary or pharmacological agents. << Less

  Phytochemistry 56:5-51(2001) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli.

  Marillia E.F., MacPherson J.M., Tsang E.W., Van Audenhove K., Keller W.A., GrootWassink J.W.

  A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library ... >> More

  A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase. << Less

  Physiol Plant 113:176-184(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis.

  Grubb C.D., Zipp B.J., Ludwig-Mueller J., Masuno M.N., Molinski T.F., Abel S.

  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 demonstra ... >> More

  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. << Less

  Plant J. 40:893-908(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.