omega-hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possibleinvolvement in plant defence

The C-18 fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxyst earic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa, The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9 ,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer, Both the 9S,10R and 9R,10S enant iomers were incubated separately. We determined respective K-m and V-max va lues of 1.2 +/- 0.1 muM and 19.2 +/- 0.3 nmol/min per nmol of cytochrome P4 50 for the 9R,10S enantiomer and of 5.9 +/- 0.1 muM and 20.2 +/- 1.0 nmol/m in per nmol of cytochrome P450 for the 9S,10R enantiomer, This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially f ormed in V. sativa microsomes. Cutin analysis of V. sativa seedlings reveal ed that it is mainly constituted of derivatives of palmitic acid, a C-16 fa tty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10- epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.