出版社：American Society for Biochemistry and Molecular Biology
摘要：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 10 R -DOX and epoxy alcohol synthase (EAS) activities. This enzyme, 10 R -DOX-EAS, transformed 18:2 n -6 sequentially to 10( R )-hydroperoxy-8( E ),12( Z )-octadecadienoic acid (10 R -HPODE) and to 12 S (13 R )-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 18:2 n -6, suggesting antarafacial hydrogen ion and oxygenation. Experiments with 18O2 and 16O2 gas confirmed that the epoxy alcohol was formed from 10 R -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. 10 R -DOX-EAS is the first member of a novel subfamily of DOX-CYP fusion proteins of devastating plant pathogens.
关键词：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
