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.

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 seedling 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.

Stereochemistry of oxidized fatty acids generated during catalytic oxygenation of lauric acid and unsaturated analogs by plant microsomes.

The capacity of microsomes from aminopyrine-induced Jerusalem artichoke (Helianthus tuberosus L.) to oxidize saturated and unsaturated fatty acids has been investigated using lauric acid and a series of unsaturated lauric acid analogs (7-, 8-, 9- and 10-dodecenoic acids) as radiolabeled substrates. In the presence of NADH, lauric acid was mono-hydroxylated principally at carbon 9. Steric analysis of this product showed a low enantiomeric excess of 28%. Mono-hydroxylated and mono-epoxidated reaction products were formed from the unsaturated analogs. The epoxidation/hydroxylation ratio was related to the position of the double bond in the aliphatic chain. The oxidation of 7-dodecenoic acid (7-DDNA) and 10-DDNA produced mainly 9-hydroxy-7-DDNA and 9-hydroxy-10-DDNA plus minor amounts of 7,8-epoxy- or 10,11-epoxylauric acid, respectively. In contrast, 8- and 9-DDNAs yielded essentially 8,9-epoxy- and 9,10-epoxylauric acids and smaller amounts of 10-hydroxy-9-DDNA and 8-hydroxy-9-DDNA, respectively. The optical purity and the absolute configuration of the major metabolites were investigated. Epoxidation of Z 8-DDNA and Z 9-DDNA occurs with high enantiomeric excesses. When the double bond was in the Z configuration, (8S,9R)/(8R,9S) 8,9-epoxylauric acid (93/7) or (9R,10S)/(9S,10R) 9,10-epoxylauric acid (89/11) were produced. In contrast, when the double bond was in the E configuration, steric analysis showed an enantiomeric ratio of 52/48 for E 8,9-epoxide and of 59/41 for E 9,10-epoxide. Z 7-DDNA led to the formation of 98% of the 9(S)-hydroxy-Z 7-DDNA enantiomer, while 9-hydroxy-Z 10-DDNA derived from Z 10-DDNA was 35% (R) and 65% (S).

Subterminal hydroxylation of lauric acid by microsomes from a marine fish.

Microsomes from the liver of sea bass (Dicentrarchus labrax) were shown to hydroxylate lauric acid at subterminal positions. The cytochrome P-450 system converted lauric acid to several mono-hydroxylated metabolites including omega-1 hydroxylaurate, which was the major metabolite (44% of total products). In addition, omega-2, omega-3, omega-4 and a small amount (2.3%) of omega hydroxylaurates were found. Reaction products were identified using thin-layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS). Oxidation reactions were dependent upon O2 and NADPH, and did not occur with boiled microsomes or in the presence of a mixture of CO/O2. Hydroxylation proceeded linearly up to 20 min at 28 degrees C for protein concentrations below 380 micrograms. Treatment of fish with benzo(a)pyrene (BP) (20 mg/kg) drastically increased xenobiotic metabolism (ECOD, EROD and BPMO activities), but no difference in laurate hydroxylase activity was observed between untreated and treated fish. Starvation strongly enhanced laurate hydroxylase activity, and resumption of feeding reduced by half this increase of activity. In all of the experiments we did not observe any modification of the regioselectivity of lauric acid hydroxylation by this microsomal in-chain hydroxylating system. We suggest that cytochrome P-450 enzymes involved in lauric acid and xenobiotics metabolism are regulated independently.