Document Detail


Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition.
MedLine Citation:
PMID:  23150922     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 catalyzes the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defense against pests and pathogens. We found that mutation of the highly conserved serine 284 to leucine (wei9-1) caused only very mild morphological and metabolic phenotypes, in dramatic contrast to 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 modeling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and kcat, sparking an interest in the kinetic behavior 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.
Authors:
Jakub Kopycki; Elisabeth Wieduwild; Janine Kohlschmidt; Wolfgang Brandt; Anna N Stepanova; Jose M Alonso; M Soledade C Pedras; Steffen Abel; C Douglas Grubb
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-15
Journal Detail:
Title:  The Biochemical journal     Volume:  -     ISSN:  1470-8728     ISO Abbreviation:  Biochem. J.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-15     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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