Function and substrate specificity of the gibberellin 3β-hydroxylase encoded by the Arabidopsis GA4 gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [C-14]gibberellin (GA)(9) and [C-14]GA(20) to radiolabeled GA(4) and GA(1), respectively, thereby confirming that GA4 encodes a GA 3 beta-hydroxylase. GA, was the preferred substrate, with a Michaelis value of 1 mu M compared with 15 mu M for GA(20). Hydroxylation of these GAs was regiospecific, with no indication of 2 beta-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other CA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA(12)-aldehyde, GA(12), GA(19), GA(25), GA(53), or GA(44) as the open lactone (20-hydroxy-GA(53)), whereas GA(15), GA(24), and GA(44) were hydroxylated to GA(37), GA(36), and GA(38), respectively. The open lactone of GA(15) (20-hydroxy-GA,,) was hydroxylated but less efficiently than GA(15). In contrast to the free acid, GA(25) 19,20-anhydride was 3 beta-hydroxylated to give GA(13). 2,3-Didehydro-GA(9), and CA(5) were converted by recombinant GA4 to the corresponding epoxides 2,3-oxide-GA(9) and GA(6).