Epoxy alcohol synthase of the rice blast fungus represents a novel subfamily of dioxygenase-cytochrome P450 fusion enzymes
2014 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 55, no 10, 2113-2123 p.Article in journal (Refereed) Published
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 O-18(2) and O-16(2) 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 Asn(965) 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.
Place, publisher, year, edition, pages
2014. Vol. 55, no 10, 2113-2123 p.
enzymology/enzyme mechanisms, fatty acid/oxidation, mass spectrometry, oxidized lipids, protein kinases/protein kinase A, heme peroxidase, linoleate diol synthase, mutagenesis/site-specific, oxylipin/biosynthesis
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-236055DOI: 10.1194/jlr.M051755ISI: 000342925400015OAI: oai:DiVA.org:uu-236055DiVA: diva2:764315
FunderKnut and Alice Wallenberg Foundation, 2004.0123