Production in vitro by the cytochrome P450CYP94A1 of major C-18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies

The major C-18 cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18- trihydroxystearic acids. These compounds are also known messengers in plant -pathogen interactions. We have previously shown that their common precurso r 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vic ia sativa microsomes (Pinot, Salaun, Bosch, Lesot, Mioskowski and Durst (19 92) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chi rality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cyt ochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9, 10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding Is-hydroxy derivatives. The reactions were not cata lysed by microsomes from yeast transformed with a void plasmid or in absenc e of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxyst earic acid with microsomes of yeast expressing CYP94A1, the chirality of th e residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiome r. Both enantiomers were incubated separately and V-max/K-m values of 16 an d 3.42 ml/min per nmol of P450 for (9R,10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compar ed with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V-max/K-m of 0.33 ml/m in per nmol of P450. Our results indicate that the activity of CYP94A1 is s trongly influenced by the stereochemistry of the 9,10-epoxide and the natur e of substituents on carbons 9 and 10, with V-max/K-m values for epoxide mu ch greater than oleic acid > diol.