A non‐canonical caleosin from A rabidopsis efficiently epoxidizes physiological unsaturated fatty acids with complete stereoselectivity

In plants, epoxygenated fatty acids ( EFA s) are constituents of oil seeds as well as defence molecules and components of biopolymers (cutin, suberin). While the pleiotropic biological activities of mammalian EFA s have been well documented, there is a paucity of information on the physiological relevance of plant EFA s and their biosynthesis. Potential candidates for EFA formation are caleosin‐type peroxygenases which catalyze the epoxidation of unsaturated fatty acids in the presence of hydroperoxides as co‐oxidants. However, the caleosins characterized so far, which are mostly localized in seeds, are poor epoxidases. In sharp contrast, quantitative RT ‐ PCR analysis revealed that PXG4 , a class II caleosin gene, is expressed in roots, stems, leaves and flowers of A rabidopsis . Expressed in yeast, PXG4 encodes a calcium‐dependent membrane‐associated hemoprotein able to catalyze typical peroxygenase reactions. Moreover, we show here that purified recombinant PXG4 is an efficient fatty acid epoxygenase, catalyzing the oxidation of cis double bonds of unsaturated fatty acids. Physiological linoleic and linolenic acids proved to be the preferred substrates for PXG4 ; they are oxidized into the different positional isomers of the monoepoxides and into diepoxides. An important regioselectivity was observed; the C ‐12,13 double bond of these unsaturated fatty acids being the least favored unsaturation epoxidized by PXG4 , linolenic acid preferentially yielded the 9,10‐15,16‐diepoxide. Remarkably, PXG4 catalyzes exclusively the formation of ( R ),( S )‐epoxide enantiomers, which is the absolute stereochemistry of the epoxides found in planta . These findings pave the way for the study of the functional role of EFA s and caleosins in plants.