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Type: Journal article
Title: A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue
Author: Bottcher, C.
Dennis, E.
Booker, G.
Polyak, S.
Boss, P.
Davies, C.
Citation: PLoS One, 2012; 7(5):1-8
Publisher: Public Library of Science
Issue Date: 2012
ISSN: 1932-6203
Statement of
Christine Böttcher, Eric G. Dennis, Grant W. Booker, Steven W. Polyak, Paul K. Boss and Christopher Davies
Abstract: An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA) by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-59-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP) mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with Ki-values 17-68-fold lower than the respective Kmvalues. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5–20 mM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function.
Keywords: Vitis; Indoleacetic Acids; Indoles; Macromolecular Substances; Multienzyme Complexes; Amino Acids; DNA Primers; Adenosine Monophosphate; Chromatography, Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Molecular Structure; Kinetics; South Australia; Tandem Mass Spectrometry
Rights: Copyright: © 2012 Böttcher et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RMID: 0020119479
DOI: 10.1371/journal.pone.0037632
Appears in Collections:Molecular and Biomedical Science publications
IPAS publications

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