Gibberellin 2-Oxidases from Seedlings of Adzuki Bean (Vigna angularis) Show High Gibberellin-Binding Activity in the Presence of 2-Oxoglutarate and Co²⁺

抄録

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 2β-hydroxylation activity by converting GA₁, GA₄, GA₉, GA₂₀, GA₄-methyl ester, and 16,17-dihydro-GA₄ to the corresponding 2β-hydroxylated gibberellins, which were identified by GC/MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [³H₄]-16,17-dihydro-GA₄ to a metabolite showing an R_f value of 16,17-dihydro-GA₃₄. RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co²⁺ instead of Fe²⁺ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [³H₄]-16,17-dihydro-GA₄ to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.