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

Abstract

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.

CYP86A33-Targeted Gene Silencing in Potato Tuber Alters Suberin Composition, Distorts Suberin Lamellae, and Impairs the Periderm's Water Barrier Function

“…Of note is that several authors point out that substrates other than free fatty acids, such as fatty acids activated, linked to glycerol or to the polymer, could be the natural substrates of CYP86A proteins (Cahoon et al, 2002;Beisson et al, 2007;Li et al, 2007;Pollard et al, 2008). Moreover, the possibility that CYP86A33 could also catalyze the complete oxidation of the methyl to the carboxyl group, as reported for CYP94A5 (Le Bouquin et al, 2001) and CYP94C1 (Kandel et al, 2007), cannot be excluded.…”

Apoplastic Diffusion Barriers in Arabidopsis

“…It remains to be determined whether this reduction is a consequence of the depletion of ω-hydroxyacids, the direct precursor of α,ω-DCA, or the consequence of a catalytic function in the subsequent oxidation of ω-hydroxyacids to α,ω-DCA, as was demonstrated for CYP94C1 (Kandel et al, 2007) and CYP94A5 (Le Bouquin et al, 2001). Alternatively α,ω-DCA can be produced by a two-step oxidation that involves a ω-hydroxyacid and a ω-oxoacid dehydrogenase activity.…”

Biosynthesis and function of polyacetylenes and allied natural products

“…In contrast to pathways where allylic and propargylic alcohols may spontaneously eliminate water, elimination of either a primary or secondary alcohol would presumably necessitate a specific dehydratase to provide a thermodynamically less favored monosubstituted alkene. In a case potentially relevant to the formation of terminal acetylenic C 9 -alkamides in Heliantheae, CYP94A5 from tobacco catalyzed the ω-hydroxylation of C 12 -C 18 saturated and unsaturated fatty acids, with a preference for 9,10-epoxystearate, and the expressed enzyme was capable of oxidizing the terminal methyl group to a carboxylic acid in yeast microsomes [180]. α, β-Acetylenic carboxylic acids are well known to readily decarboxylate non-enzymatically leading to terminal acetylenes and this process is believed to occur in numerous instances (e.g., oxidative conversion of 3G to 3F in Fig.…”