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

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combin ed use of an improved isolation medium and jasmonic acid-induced etiol ated seedlings of Sinapis alba L. An 8-fold induction of de novo biosy nthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obt ained in etiolated S. alba seedlings upon treatment with jasmonic acid . Formation of inhibitory glucosinolate degradation products upon tiss ue homogenization was prevented by inactivation of myrosinase by addit ion of 100 mM ascorbic acid to the isolation buffer. The biosynthetica lly active microsomal enzyme system converted L-tyrosine into p-hydrox yphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The K-m and V-max values of the enzyme system were 346 mu M 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 biosyn thesis of glucosinolates has been the subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 b y photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous Cytochrome P450 inhibitors confirms the involvement of cy tochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oxim es in the biosynthesis of glucosinolates and cyanogenic glycosides.