Received February 23, 2004
Returned for revision March 26, 2004
Accepted March 26, 2004
16,17-Dihydro Gibberellin A5 Competitively Inhibits a Recombinant Arabidopsis GA 3
-Hydroxylase Encoded by the GA4 Gene
Rong Zhou , Min Yu , and Richard P. Pharis *
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
* Corresponding author; email: rpharis{at}ucalgary.ca.
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 (K
m) values of 1.25 and 10 µ
M, 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 K
m of the enzyme reaction, not the V
max, giving a dissociation constant of the enzyme-inhibitor complex (K
i) of 70 µ
M. 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.
