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.
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February 2013
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Research Article|
January 24 2013
Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition Available to Purchase
Jakub Kopycki;
Jakub Kopycki
*Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
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Elizabeth Wieduwild;
Elizabeth Wieduwild
*Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
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Janine Kohlschmidt;
Janine Kohlschmidt
†Department of Applied Biosciences and Process Engineering, Anhalt University of Applied Sciences, 06366 Köthen, Germany
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Wolfgang Brandt;
Wolfgang Brandt
‡Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany
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Anna N. Stepanova;
Anna N. Stepanova
§Department of Genetics, North Carolina State University, Raleigh, NC 27695, U.S.A.
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Jose M. Alonso;
Jose M. Alonso
§Department of Genetics, North Carolina State University, Raleigh, NC 27695, U.S.A.
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M. Soledade C. Pedras;
M. Soledade C. Pedras
¶Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5C9
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Steffen Abel;
Steffen Abel
*Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
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C. Douglas Grubb
C. Douglas Grubb
1
*Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
1To whom correspondence should be addressed (email: [email protected]).
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Publisher: Portland Press Ltd
Received:
September 07 2012
Revision Received:
November 06 2012
Accepted:
November 15 2012
Accepted Manuscript online:
November 15 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 450 (1): 37–46.
Article history
Received:
September 07 2012
Revision Received:
November 06 2012
Accepted:
November 15 2012
Accepted Manuscript online:
November 15 2012
Citation
Jakub Kopycki, Elizabeth Wieduwild, Janine Kohlschmidt, Wolfgang Brandt, Anna N. Stepanova, Jose M. Alonso, M. Soledade C. Pedras, Steffen Abel, C. Douglas Grubb; Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition. Biochem J 15 February 2013; 450 (1): 37–46. doi: https://doi.org/10.1042/BJ20121403
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