Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA(14) and GA(4), respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3 beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C-19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA(12) (C-20 methyl) with cultures of the mutant suspended in [O-18]H2O or maintained under an atmosphere of [O-18]O-2:N-2 (20:80), that GA(15) (C-20 alcohol) and GA(24) (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2. (c) 2007 Elsevier Ltd. All rights reserved.