Gibberellin 2-Oxidases from Seedlings of Adzuki Bean (Vigna angularis) Show High Gibberellin-Binding Activity in the Presence of 2-Oxoglutarate and Co2+
Bioscience, biotechnology and biochemistry
Five full-length cDNA encoding gibberellin 2-oxidases, VaGA2oxA1, VaGA2oxA2, VaGA2oxB1, VaGA2oxB2, and VaGA2oxB3, were cloned from etiolated adzuki bean (Vigna angularis cv. Dainagon) seedlings, and their enzymatic characteristics were examined using recombinant enzymes fused with glutathione S-transferase (GST). Recombinant VaGA2oxA1 (rVaGA2oxA1) and rVaGA2oxA2 showed 2-hydroxylation activity by converting GA 1 , GA 4 , GA 9 , GA 20 , GA 4 -methyl ester, and 16,17-dihydro-GA 4 to the
... ing 2-hydroxylated gibberellins, which were identified by GC/ MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [ 3 H 4 ]-16,17dihydro-GA 4 to a metabolite showing an R f value of 16,17-dihydro-GA 34 . RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co 2þ instead of Fe 2þ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [ 3 H 4 ]-16,17-dihydro-GA 4 to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.