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and the ‡ ‡IACR-Rothamsted, Harpenden, AL5 2JQ, United Kingdom CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450 monooxygenase. We searched the genome of A. thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides. By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli. The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a "P450mox" that converted oximes into toxic compounds that the plant detoxified into glucosinolates.Glucosinolates are naturally occurring amino acid-derived S-glucosides of thiohydroximate-O-sulfonates. They co-occur with endogenous thioglucosidases called myrosinases that upon tissue damage hydrolyze glucosinolates into a wide range of degradation products such as e.g. isothiocyanates, nitriles, and thiocyanates. Glucosinolates (or rather their degradation products) are involved in plant defense and constitute characteristic flavor compounds and cancer-preventive agents in Brassica vegetables.The biosynthetic pathway from precursor amino acid to the core glucosinolate structure has been well studied, and many of the intermediates are known, including oximes, thiohydroximic acids, and desulfoglucosinolates (1, 2). Recently, it has been shown that cytochromes P450 belonging to the CYP79 family catalyze the conversion of amino acids to oximes (3-7). Little is known about the formation of thiohydroximic acids from oximes. The remaining part of the pathway for the core structure involves a UDP-glucose:thiohydroximic acid glucosyltransferase and a sulfotransferase (for review, see Ref.2).It has been proposed that aci-nitro compounds are intermediates in the conversion of oximes to thiohydroximic acids (8). This was supported by isolation of 1-nitro-2-phenylethane from Tropaeolum majus shoots and by in vivo conversion of phenylacetaldoxime into 1-nitro-2-phenylethane and of 1-nitro-2-[1,2-14 C]phenylethane into benzylglucosinolate (9). The aci-nitro is proposed to be conjugated with a sulfur donor to produce an S-alkyl thiohydroximate, possibly by a glutathione S-transferase (2). Biochemical studies indicate that the S-alkyl thiohydroximate is subsequently hydrolyzed to the thiohydroximic acid by a C-S lyase (10).Glucosinolates are related to cyanogenic glucosides because both...

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.

An in vitro system that converts phenylalanine to phenylacetaldoxime in the biosynthesis of the glucosinolate glucotropaeolin has been established in seedlings of Tropaeolum majus L. exposed to the combined treatment of jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold induction, compared with untreated, dark-grown seedlings, of de novo biosynthesis measured as incorporation of radioactively labeled phenylalanine into glucotropaeolin. Formation of the inhibitory degradation product benzylisothiocyanate during tissue homogenization was prevented by inactivation of the thioglucosidase myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. This allowed the isolation of a biosynthetically active microsomal preparation from the induced T. majus plant material. The enzyme, which catalyzes the conversion of phenylalanine to the corresponding oxime, was sensitive to cytochrome P450 inhibitors, indicating the involvement of a cytochrome P450 in the biosynthetic pathway. It has previously been shown that the oxime-producing enzyme in the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is dependent on cytochrome P450, whereas the oxime-producing enzymes in Brassica species have been suggested to be flavin monooxygenases or peroxidase-type enzymes. The result with T. majus provides additional experimental documentation for a similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glucosides.