SEPARATION AND CHARACTERIZATION OF 3 2-OXOGLUTARATE-DEPENDENT DIOXYGENASES FROM CUCURBITA-MAXIMA L ENDOSPERM INVOLVED IN GIBBERELLIN BIOSYNTHESIS

Three enzymes of the gibberellin (GA) biosynthetic pathway, a 7-oxidase, a 20-oxidase and a 3 beta-hydroxylase, were partially purified from Cucurbita maxima endosperm by ammonium sulfate precipitation, gel-filtration and anion-exchange chromatography. The enzyme activities, which were assayed by the oxidation of GA(12)-aldehyde to GA(12), of GA(12) to GA(15) (and GA(24)) and of GA(15) to GA(37), respectively, were completely separated from each other. The apparent molecular masses as estimated by gel-filtration high-performance liquid chromatography were 34.5 kDa for the 7-oxidase, 44.5 kDa for the 20-oxidase and 58 kDa for the 3 beta-hydroxylase. The Michaelis-Menten constants (K-m) were 8.6 mu M, 0.15 mu M and 8.7 mu M for the respective substrates. All three enzymes had properties typical of 2-oxoglutarate-dependent dioxygenases. 2-Oxoglutarate was essential for activity and served as a co-substrate, giving K-m values of 6.1 mu M, 91 mu M and 41 mu M with the 7-oxidase, 20-oxidase and 3 beta-hydroxylase, respectively. Furthermore, 2-oxo[5-C-14]glutarate was oxidised stoichiometrically to [C-14]succinate when the GA-substrates were oxidised to their respective products, and the 1:1 ratio was maintained under different oxygen concentrations. Approximately equimolar amounts of (CO2)-C-14 were released from 2-oxo[1-C-14]glutarate when GA(12) was oxidised to GA(15/24) by the 20-oxidase. A crude enzyme preparation containing all three enzyme activities (and a 2 beta-hydroxylase) converted GA(12)-aldehyde to [O-18(2)]GA(4) and [O-18(5)]GA(43) under O-18(2), showing that all O-atoms incorporated after GA(12)-aldehyde originate from O-2. Accordingly, the reaction rates were near zero under anaerobic conditions, although very low concentrations of O-2 sufficed to sustain the reactions. Both Fe2+ and dithiothreitol stimulated the enzyme activities strongly, but if they were added together, catalase was needed to prevent inhibition. The pH dependence showed two opposite trends; the 7-oxidase was most active at pH 6 and below, whereas the other enzymes were maximally active above pK 6.5.