Abstract

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Highlights

  • IntroductionThe fatty acid peroxide-metabolizing P450s are by far the better known and include CYP5 (thromboxane synthase) and CYP8A (prostacyclin synthase) in animals [11], and the entire family of CYP74 in plants [12]

  • The fatty acid peroxide-metabolizing P450s are by far the better known and include CYP5 and CYP8A in animals [11], and the entire family of CYP74 in plants [12]

  • Expression, and Purification of the Mini-catalase/ Lipoxygenase Fusion Protein—BLAST searches using the catalase-related allene oxide synthase domain of Plexaura homomalla identified a good match (E value, 3e-30; 29% amino acid identity) on the main chromosome of A. marina MBIC11017, a hypothetical protein of 805 amino acids designated as ABW27596

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Summary

Introduction

The fatty acid peroxide-metabolizing P450s are by far the better known and include CYP5 (thromboxane synthase) and CYP8A (prostacyclin synthase) in animals [11], and the entire family of CYP74 in plants [12]. The known activities include AOS in Plexaura homomalla and other marine corals (with a different specificity for fatty acid hydroperoxide compared with the plant P450 AOS) [15, 16], and the unique bicyclobutane synthase and other allylic epoxide synthase activities of the enzyme in the cyanobacterium Anabaena PCC-7120 [17, 18]. The underlying chemistry of the fatty acid hydroperoxide transformations by the specialized P450 and catalase-related hemoproteins can be written as purely free radical in character, or ionic, or with facets of both (19 –22). BLAST searches of the A. marina genome reveal three individual LOX sequences and the presence of DNA encoding a putative fusion protein of lipoxygenase and catalase-related hemoprotein, the topic of this report. We find an unexpected by-product in the reaction with one particular fatty acid (C18.2␻6); the partial incorporation of 18O from water in a newly formed hydroxyl group has implications related to the mechanism of hydroperoxide transformation

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