CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydr oxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was exp ressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabi dopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes . When incubated for 10 min in presence of NADPH with microsomes of recombi nant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic param eters of the reaction were K-m,K-app = 0.9 +/- 0.2 mum and V-max,V-app = 27 +/- 1 nmol.min(-1).nmol(-1) P450. Increasing the incubation time to 1 h le d to the formation of a compound identified by GC/MS as 9,10-epoxy-octadeca n-1,18-dioic acid. The diacid was also produced in microsomal incubations o f 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incub ations 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 complet e set of reactions oxidizing a terminal methyl group to the corresponding c arboxyl. This new fatty acid hydroxylase is enantioselective: after incubat ion 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 a lso catalyzed the omega -hydroxylation of saturated and unsaturated fatty a cids with aliphatic chain ranging from C12 to C18.