title: Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases.
abstract: The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, 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. 3beta-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 C19-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 GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (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.
authors.affiliation: Laboratorio de Bioorgánica, Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
authors.forename: Claudia
authors.initials: C
authors.lastname: Troncoso
authors.forename: José
authors.initials: J
authors.lastname: Cárcamo
authors.forename: Peter
authors.initials: P
authors.lastname: Hedden
authors.forename: Bettina
authors.initials: B
authors.lastname: Tudzynski
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Rojas
mesh_terms: D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005670:Fusarium / Q000201:enzymology* / Q000378:metabolism; D005875:Gibberellins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008968:Molecular Conformation; D009154:Mutation; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D005875:Gibberellins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases