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Structure type: monomer
Trivial name: 3-methylindolyl glucosinolate

• Article ID: 11211
  Grubb CD, Zipp BJ, Ludwig-Müller J, Masuno MN, Molinski TF, Abel S "Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis" - Plant Journal: for Cell and Molecular Biology 40 (2004) 893-908
  

  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 μm) and UDP-glucose (approximately 50 μm) 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.

  glucosyltransferase, Thioglycosides, Secondary metabolism, Auxin, glucosinolate pathway, thiohydroximates

  Publication DOI: 10.1111/j.1365-313X.2004.02261.x
  Journal NLM ID: 9207397
  Publisher: Oxford: Blackwell Scientific Publishers and BIOS Scientific Publishers for the Society for Experimental Biology
  Correspondence: sabel@ucdavis.edu
  Institutions: Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA, Department of Plant Sciences, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA, Institut für Botanik, Technische Universität Dresden, Zellescher Weg 22, D-01062, Germany
  Methods: GC-MS, biological assays, HPLC, enzymatic assay, genetic manipulations
  
   
  
  Arabidopsis thaliana
  CSDB ID 60828 (all data & tools)

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Structure type: monomer
Trivial name: 3-indolylmethyl glucosinolate

• Article ID: 11212
  Kopycki J, Wieduwild E, Kohlschmidt J, Brandt W, Stepanova AN, Alonso JM, Pedras MSC, Abel S, Grubb CD "Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition" - Biochemical Journal 450 (2013) 37-46
  

  Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.

  glycosyltransferase, glucosinolate, ordered binding, product inhibition, UDP-glycosyltransferase (UGT)

  Publication DOI: 10.1042/BJ20121403
  Journal NLM ID: 2984726R
  Publisher: London, UK : Published by Portland Press on behalf of the Biochemical Society
  Institutions: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany, Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany, Department of Applied Biosciences and Process Engineering, Anhalt University of Applied Sciences, 06366 Köthen, Germany, Department of Genetics, North Carolina State University, Raleigh, NC 27695, U.S.A., Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5C9
  Methods: biological assays, enzymatic assay, UPLC, genetic manipulations, molecular modelling
  
   
  
  Arabidopsis thaliana
  CSDB ID 60834 (all data & tools)