Partial purification and characterization of gibberellin 3β-hydroxylase from immature seeds of phaseolus vulgaris l.

Abstract

Gibberellin 3/β-hydroxylase,a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of GA20 to GA1, was purified 313-fold from immature seeds of Phaseolus vulgaris L. The mol wt of the enzyme was estimated to be 42,000 by gel filtration HPLC and SDS-polyacrylamide gel electrophoresis. The enzyme exhibited maximum activity at pH 7.7. The Km values for [2,3-3H]GA20 and [2,3-3H]GA, were 0.29μu and 0.33 μm, respectively. The enzyme requires 2-oxoglutarate as a cosubstrate; the Km value for 2-oxoglutarate was 250μM using [3H]- GA20 as a substrate. Fe2+ and ascorbate significantly activated the enzyme at all purification steps, while catalase and BSA activated the purified enzyme only. The enzyme was inhibited by divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. 3β-Hydroxylation of [3H]- GA20 was also inhibited by non-radioactive GA5, GA9,GA15, GA20 and GA44. The possible site of 3β-hydroxylation in gibberellin biosynthesis is discussed in terms of the substrate specificity of partially purified gibberellin 3β-hydroxylase. © 1988 The Japanese Society of Plant Physiologists (JSPP).