Separation and characterisation of three 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-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 GA₁₂-aldehyde to GA₁₂, of GA₁₂ to GA₁₅ (and GA₂₄) and of GA₁₅ to GA₃₇, 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-hydroxylase. The Michaelis-Menten constants (K _m) were 8.6 M, 0.15M and 8.7 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 M, 91 M and 41 M with the 7-oxidase, 20-oxidase and 3-hydroxylase, respectively. Furthermore, 2 oxo5-¹⁴C]glutarate was oxidised stoichiometrically to ¹⁴C]succinate when the GA-substrates were oxidised to their respective products, and the 11 ratio was maintained under different oxygen concentrations. Approximately equimolar amounts of ¹⁴CO₂ were released from 2-oxo1-¹⁴C]glutarate when GA₁₂ was oxidised to GA_15/24 by the 20-oxidase. A crude enzyme preparation containing all three enzyme activities (and a 2-hydroxylase) converted GA₁₂-aldehyde to ¹⁸O₂]GA₄ and ¹⁸O₅]GA₄₃ under ¹⁸O₂, showing that all O-atoms incorporated after GA₁₂-aldehyde originate from O₂. Accordingly, the reaction rates were near zero under anaerobic conditions, although very low concentrations of O₂ 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 pH 6.5.Abbreviations BSA bovine serum albumin - GA_n gibberellin A_n - DTT dithiothreitol - GC-MS combined gas chromatography-mass spectrometry - MeTMSi methyl ester trimethylsilyl ether We thank Mr. Keith Hall (Long Ashton) for assistance with the oxygen concentration measurements and Mrs. Gudrun Bodtke (Göttingen) and Mrs. Brigitte Schattenberg (Göttingen) for able technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft, Germany, and the Agricultural and Food Research Council, UK, and by an Academic Research Collaboration award jointly from the Deutsche Akademische Austauschdienst (DAAD) and the British Council.