Gibberellin 2-oxidases from seedlings of adzuki bean (Vigna angularis) show high gibberellin-binding activity in the presence of 2-oxoglutarate and Co2+.
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 2beta-hydroxylation activity by converting GA1, GA4, GA9, GA20, GA4-methyl ester, and 16,17-dihydro-GA4 to the corresponding 2beta-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 Rf 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.
Park
Seung-Hyun
SH
Department of Applied Biological Chemistry, University of Tokyo, Japan.
Nakajima
Masatoshi
M
Sakane
Masayuki
M
Xu
Zheng-Jun
ZJ
Tomioka
Katsushi
K
Yamaguchi
Isomaro
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology*; D017931:DNA Primers; D007887:Fabaceae / Q000201:enzymology* / Q000254:growth & development; D005875:Gibberellins / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D036226:Seedlings / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005875:Gibberellins; D007656:Ketoglutaric Acids; D003035:Cobalt; D006899:Mixed Function Oxygenases; C121463:gibberellin 2-dioxygenase