title: Characterization and Solubilization of Kaurenoic Acid Hydroxylase from Gibberella fujikuroi.
abstract: A key step in gibberellin biosynthesis is the conversion of ent-kaurenoic acid to ent-7[alpha]-hydroxykaurenoic acid, mediated by the enzyme kaurenoic acid hydroxylase. A cell-free system obtained from Gibberella fujikuroi (Saw.) Wr. was used to characterize kaurenoic acid hydroxylase activity. Microsomal preparations from disrupted fungal cells, in the presence of O2 and NADPH, converted [17-14C]ent-kaurenoic acid to oxidation products that were separated by high-performance liquid chromatography and identified as ent-7[alpha]-hydroxykaurenoic acid and gibberellin A14 by combined gas chromatography-mass spectrometry. Flavin adenine dinucleotide and the chloride salts of several monovalent cations stimulated the conversion of ent-kaurenoic acid to these products, whereas CO and a number of known inhibitors of cytochrome P-450-dependent reactions, including paclobutrazol, tetcyclacis, BAS 111.W, flurprimidol, triarimol, metyrapone, and 1-phenylimida-zole, significantly reduced kaurenoic acid hydroxylase activity. Kaurenoic acid hydroxylase was solubilized from fungal microsomes by treatment with 1 M KCl. The properties of the enzyme noted above suggest that kaurenoic acid hydroxylase from G. fujikuroi is a cytochrome P-450-dependent monooxygenase.
authors.affiliation: Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 (J.C.J., R.C.C.).
authors.forename: J. C.
authors.initials: JC
authors.lastname: Jennings
authors.forename: R. C.
authors.initials: RC
authors.lastname: Coolbaugh
authors.forename: D. A.
authors.initials: DA
authors.lastname: Nakata
authors.forename: C. A.
authors.initials: CA
authors.lastname: West