Characterization of kaurenoic acid hydroxylase from Gibberella fujikuroi
Abstract
An essential reaction in the biosynthesis of all gibberellins (GAs) is the conversion of ent-kaurenoic acid to ent-7$\alpha$-hydroxykaurenoic acid, mediated by kaurenoic acid hydroxylase. A GA-producing fungus, Gibberella fujikuroi (Saw.) Wr., was used to characterize cell-free kaurenoic acid hydroxylase activity. Microsomal preparations, in the presence of O$\sb2$ and NADPH, converted ent-kaurenoic acid to ent-7$\alpha$-hydroxykaurenoic acid. FAD and monovalent chloride salts stimulated this conversion, whereas CO, cytochrome c, and several cytochrome P450 inhibitors reduced enzyme activity. Kaurenoic acid hydroxylase was also solubilized from fungal microsomes by treatment with 1 M KCl. These properties suggest that the hydroxylase is a cytochrome P450 monooxygenase. Subtractive hybridization was used to attempt to isolate cDNAs corresponding to transcripts involved in the biosynthesis of GAs, specifically those related to kaurenoic acid hydroxylase. Cultures of G. fujikuroi grown in the presence of 84 $\mu$M AMO-1618 (an inhibitor of ent-kaurene synthesis) showed reduced levels of GA$\sb3$ in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. The level of hydroxylase activity from AMO-1618-treated cultures was induced above the untreated level by growing cultures in the presence of 350 $\mu$M ent-kaurene. A mutant strain of G. fujikuroi (B1-41a), blocked prior to kaurenoic acid hydroxylase, was also deficient in GA$\sb3$ production and hydroxylase activity. Since transcripts related to GA biosynthesis might be decreased in AMO-1618-treated and mutant cultures, a subtractive hybridization procedure was used to isolate cDNA fragments for messages that were more abundant in wild-type than treated or mutant cultures. Subtractions were performed between cDNAs from wild-type and AMO-1618-treated cultures, and wild-type and mutant cultures. A wild-type cDNA library was screened with both sets of subtraction products, and four cDNA clones were purified and partially characterized. One cDNA hybridized to transcripts that were down-regulated in AMO-1618-treated cultures and undetected in mutant cultures. Three clones, isolated from the wild-type/mutant subtractive hybridization, corresponded to transcripts that were more abundant in wild-type and treated cultures than in mutant cultures.
Degree
Ph.D.
Advisors
Coolbaugh, Purdue University.
Subject Area
Botany
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