ISOLATION AND RECONSTITUTION OF CYTOCHROME P450OX AND IN-VITRO RECONSTITUTION OF THE ENTIRE BIOSYNTHETIC-PATHWAY OF THE CYANOGENIC GLUCOSIDE DHURRIN FROM SORGHUM

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside ta-D-glucopyranosyloxy-( S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsome s prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moe nch). P450ox was solubilized using nonionic detergents, and isolated b y ion-exchange chromatography, Triton X-114 phase partitioning, and dy e-column chromatography. P450ox has an apparent molecular mass of 55 k D, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it conta ins the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatid ylcholine identified P450ox as a multifunctional P450 catalyzing dehyd ration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hy droxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extre mely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diph osphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydrox ylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The cata lysis of the conversion of Tyr into nitrile by two multifunctional P45 0s explains why all intermediates in this pathway except (Z)-oxime are channeled.