Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation of the terminal olefin by plant cytochrome P-450

Abstract

The study of the stereochemistry and regioselectivity of plant fatty acid hydroxylases is hampered by the difficulty to purify plant cytochrome P-450 enzymes. To provide an alternative, we have now defined an experimental plant system which expresses only one hydroxylase activity towards lauric acid: microsomes from clofibrate-induced Vicia sativa seedlings hydroxylate this fatty acid exclusively at the methyl terminus. To explore the catalytic capabilities of this laurate oxidase, a series of 1-¹⁴C-radiolabeled unsaturated lauric acid analogs (7-, 8-, 9-, 10- and 11-dodecenoic acids) were synthesized. Microsomes from clofibrate induced Vicia sativa seedlings catalyzed the omega-oxidation of the lauric acid analogs in the presence of O₂ and NADPH. The cis and trans forms of the four in-chain unsaturated analogs of lauric acid were 12-hydroxylated with similar efficiency. The terminal olefin was readily converted to the epoxide with only marginal autocatalytic inactivation of the enzyme. The formation of each metabolite was inhibited to the same extend when microsomes were incubated in presence of carbon monoxide or a suicide-substrate for omega LAH, suggesting that a single cytochrome P-450 isoenzyme from Vicia sativa microsomes is able to omega hydroxylate lauric acid and in-chain unsaturated analogs, and to epoxygenate 11-dodecenoic acid.