A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside β-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-α--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.

A full length cDNA encoding a new cytochrome P450‐dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10‐epoxystearic acid was converted into one major metabolite identified by GC/MS as 18‐hydroxy‐9,10‐epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 ± 0.2 µm and Vmax,app = 27 ± 1 nmol·min−1·nmol−1 P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10‐epoxy‐octadecan‐1,18‐dioic acid. The diacid was also produced in microsomal incubations of 18‐hydroxy‐9,10‐epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10‐epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the ω‐hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.

Obtusifoliol 14 alpha-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14 alpha-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14 alpha-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14 alpha-demethylases are orthologous enzymes. The sterol 14 alpha-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14 alpha-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14 alpha-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH-cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14 alpha-demethylase catalyses the 14 alpha-demethylation of obtusifoliol to 4 alpha-methyl-5 alpha-ergosta-8, 14,24(28)-trien-3 beta-ol as evidenced by GC-MS. The isolation of a cDNA clone encoding the plant sterol 14 alpha-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14 alpha-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14 alpha-demethylases.

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