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A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.
PLoS One. 2012; 7(5):e37632.Plos

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-5'-[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 K(i)-values 17-68-fold lower than the respective K(m)-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5-20 µM 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.

Authors+Show Affiliations

CSIRO Plant Industry, Glen Osmond, South Australia, Australia. christine.bottcher@csiro.auNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22649546

Citation

Böttcher, Christine, et al. "A Novel Tool for Studying Auxin-metabolism: the Inhibition of Grapevine Indole-3-acetic Acid-amido Synthetases By a Reaction Intermediate Analogue." PloS One, vol. 7, no. 5, 2012, pp. e37632.
Böttcher C, Dennis EG, Booker GW, et al. A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue. PLoS One. 2012;7(5):e37632.
Böttcher, C., Dennis, E. G., Booker, G. W., Polyak, S. W., Boss, P. K., & Davies, C. (2012). A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue. PloS One, 7(5), e37632. https://doi.org/10.1371/journal.pone.0037632
Böttcher C, et al. A Novel Tool for Studying Auxin-metabolism: the Inhibition of Grapevine Indole-3-acetic Acid-amido Synthetases By a Reaction Intermediate Analogue. PLoS One. 2012;7(5):e37632. PubMed PMID: 22649546.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue. AU - Böttcher,Christine, AU - Dennis,Eric G, AU - Booker,Grant W, AU - Polyak,Steven W, AU - Boss,Paul K, AU - Davies,Christopher, Y1 - 2012/05/23/ PY - 2012/03/20/received PY - 2012/04/27/accepted PY - 2012/6/1/entrez PY - 2012/6/1/pubmed PY - 2012/10/10/medline SP - e37632 EP - e37632 JF - PloS one JO - PLoS One VL - 7 IS - 5 N2 - 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-5'-[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 K(i)-values 17-68-fold lower than the respective K(m)-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5-20 µM 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/22649546/A_novel_tool_for_studying_auxin_metabolism:_the_inhibition_of_grapevine_indole_3_acetic_acid_amido_synthetases_by_a_reaction_intermediate_analogue_ L2 - https://dx.plos.org/10.1371/journal.pone.0037632 DB - PRIME DP - Unbound Medicine ER -