First published online June 4, 2004; 10.1104/pp.104.041509
Plant Physiology 135:1000-1007 (2004)
© 2004 American Society of Plant Biologists
DEVELOPMENT AND HORMONE ACTION
16,17-Dihydro Gibberellin A5 Competitively Inhibits a Recombinant Arabidopsis GA 3 -Hydroxylase Encoded by the GA4 Gene
Rong Zhou1,
Min Yu2 and
Richard P. Pharis*
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
Ring D-modified gibberellin (GA) A5 and A20 derivatives are structurally similar to GA20 and GA9 (the precursors to growth-active GA1 and GA4) and, when applied to higher plants, especially grasses, can reduce shoot growth with concomitant reductions in levels of growth-active GAs and increases in levels of their immediate 3-deoxy precursors. The recombinant Arabidopsis GA 3 -hydroxylase (AtGA3ox1) protein was used in vitro to test a number of ring D-modified GA structures as possible inhibitors of AtGA3ox1. This fusion protein was able to 3-hydroxylate the 3-deoxy GAs, GA9 and GA20, to GA4 and GA1, respectively, and convert the 2,3-didehydro GA, GA5, to its 2,3-epoxide, GA6. Michaelis-Menten constant (Km) values of 1.25 and 10 µM, respectively, were obtained for the GA9 and GA20 conversions. We utilized the enzyme's ability to convert GA20 to GA1 in order to test the efficacy of GA5, 16,17-dihydro GA5 (dihydro GA5), and a number of other ring D-modified GAs as inhibitors of AtGA3ox activity. For the exo-isomer of dihydro GA5, inhibition increased with the dose of dihydro GA5, with Lineweaver-Burk plots showing that dihydro GA5 changed only the Km of the enzyme reaction, not the Vmax, giving a dissociation constant of the enzyme-inhibitor complex (Ki) of 70 µM. Other ring D-modified GA derivatives showed similar inhibitory effects on GA1 production, with 16,17-dihydro GA20-13-acetate being the most effective inhibitor. This behavior is consistent with dihydro GA5, at least, functioning as a competitive substrate inhibitor of AtGA3ox1. Finally, the recombinant AtGA3ox1 fusion protein may be a useful screening tool for other effective 3-hydroxylase inhibitors, including naturally occurring ones.
1 Present address: Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N 0W9.
2 Present address: Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada S7N 0X2.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041509.
* Corresponding author; e-mail rpharis{at}ucalgary.ca; fax 403–289–9311.
Received February 23, 2004;
returned for revision March 26, 2004;
accepted March 26, 2004.
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