A
cytochrome P450, designated
P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (
oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic
glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (
dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench).
P450ox was solubilized using nonionic
detergents, and isolated by ion-exchange chromatography,
Triton X-114 phase partitioning, and
dye-column chromatography.
P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of
P450ox with NADPH-P450
oxidoreductase in
micelles of L-alpha-dilauroyl
phosphatidylcholine identified
P450ox as a multifunctional P450 catalyzing
dehydration of (Z)-
oxime to p-hydroxyphenylaceto-
nitrile (
nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to
nitrile.
P450ox is extremely labile compared with the P450s previously isolated from sorghum. When
P450ox is reconstituted in the presence of a soluble
uridine diphosphate 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-hydroxylase),
P450ox, and NADPH-P450
oxidoreductase in
lipid micelles in the presence of
uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into
nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-
oxime are channeled.