Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum

Abstract

Fusarium oxysporum is a devastating plant pathogen that oxidizes C 18 fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB-01332. Recombinant FOXB-01332 oxidized 18:2n-6 to 9 S-hydroperoxy-10(E), 12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9 S (10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9 S-DOX-AOS. The 9 S-DOX activity oxidized C18 and C 20 fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB-01332 has acidic and alcoholic residues, Glu946-Val-Leu-Ser949, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB-01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.