Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

Abstract

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

“…12). In microsomal enzyme systems isolated from S. alba, T. majus, and C. papaya, tyrosine or phenylalanine are converted to the corresponding aldoximes by cytochrome P450-dependent monooxygenases, as evidenced by photoreversible carbon monoxide inhibition and other inhibitor studies (13)(14)(15)(16). Biochemical studies with microsomal preparations from Brassica napus L. have indicated that the conversion of homophenylalanine, dihomo-, trihomo-, and tetrahomomethionine to the corresponding aldoximes is not affected by carbon monoxide and 2 P. Brown and J. Gershenzon, personal communication.…”

“…Independent biochemical studies have indicated that three different enzyme systems are involved in this step, namely cytochrome P450-dependent monooxygenases, flavincontaining monooxygenases, and peroxidases (12). The aromatic amino acids (tyrosine and phenylalanine) are converted to the corresponding aldoximes by cytochrome P450-dependent monooxygenases in microsomes isolated from Sinapis alba L., Tropaeolum majus L., and Carica papaya L. (13)(14)(15)(16). Conversion of homophenylalanine and chain elongated methionine homologues into the corresponding aldoximes by microsomal preparations of Brassica spp.…”

Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis

“…Because genes involved in Trp biosynthesis and its secondary metabolites are induced in response to pathogens (22,31,32), we tested whether CYP79B2 mRNA expression is induced in response to bacterial infection. A Northern blot was prepared from Col-0 Arabidopsis leaves that were infected for various times with either the virulent strain P. syringae pv.…”

Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis

“…In Arabidopsis, glucosinolates are biosynthesized from a variety of typical protein amino acids (methionine, tryptophan, and phenylalanine) and their chain-elongated analogs. The first step in glucosinolate biosynthesis is the oxidation of the amino group to an oxime moiety by amino acid-specific cytochrome P450 monooxygenases (Du et al, 1995;Wittstock and Halkier, 2000). After a thiohydroximate intermediate is formed, sequential transfer of glucose and sulfate residues creates the basic glucosinolate skeleton (Halkier and Du, 1997).…”