Epoxy alcohol synthase of the rice blast fungus represents a novel subfamily of dioxygenase-cytochrome P450 fusion enzymes

The genome of the rice blast fungus Magnaporthe oryzae codes for two proteins with N-terminal dioxygenase (DOX) and C-terminal cytochrome P450 (CYP) domains, respectively. One of them, MGG_13239, was confirmed as 7,8-linoleate diol synthase by prokaryotic expression. The other recombinant protein (MGG_10859) possessed prominent 10R-DOX and epoxy alcohol synthase (EAS) activities. This enzyme, 10R-DOX-EAS, transformed 18:2n-6 sequentially to 10(R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE) and to 12S(13R)-epoxy-10(R)-hydroxy-8(E)-octadecenoic acid as the end product. Oxygenation at C-10 occurred by retention of the pro-R hydrogen of C-8 of 18:2n-6, suggesting antarafacial hydrogen abstraction and oxygenation. Experiments with ¹⁸O₂ and ¹⁶O₂ gas confirmed that the epoxy alcohol was formed from 10R-HPODE, likely by heterolytic cleavage of the dioxygen bond with formation of P450 compound I, and subsequent intramolecular epoxidation of the 12(Z) double bond. Site-directed mutagenesis demonstrated that the cysteinyl heme ligand of the P450 domain was required for the EAS activity. Replacement of Asn⁹⁶⁵ with Val in the conserved AsnGlnXaaGln sequence revealed that Asn⁹⁶⁵ supported formation of the epoxy alcohol. 10R-DOX-EAS is the first member of a novel subfamily of DOX-CYP fusion proteins of devastating plant pathogens.