Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

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 18.4ω3 and 18.3ω3 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 ∼70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only ∼30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H₂¹⁸O indicated that ∼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.A diverse spectrum of signaling molecules is biosynthesized in nature from polyunsaturated fatty acids, their peroxides, and further transformations of the fatty acid peroxides. The peroxides are formed by two classes of dioxygenase enzyme. The hemoprotein dioxygenases include prostaglandin H synthase (cyclooxygenase) in animals (1), α-dioxygenase in plants (2), and several linoleate dioxygenases in fungi (3–5). The non-heme iron lipoxygenases are even more widespread, being almost ubiquitous among organisms that contain polyunsaturated fatty acids (6–8). Although further biosynthetic transformation is sometimes accomplished by an additional catalytic activity of the initiating dioxygenase (e.g. leukotriene A₄ synthase (9) or aldehyde-synthesizing hydroperoxide cleaving activity (10) among the LOX² enzymes), commonly another distinct enzyme is used to rearrange or otherwise modify the reactive fatty acid peroxide intermediate. Two hemoprotein types are found that have become specialized for this biosynthetic role: cytochrome P450s and catalase-related enzymes.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 individual CYP74 enzymes include allene oxide synthase (AOS), one of which catalyzes a key step in cyclopentenone synthesis in the jasmonate pathway, hydroperoxide lyase, divinyl ether synthase, and epoxyalcohol synthase (12, 13). The catalase-related enzymes are distinctive in that they have been found naturally as a fusion protein with the LOX enzyme that forms their hydroperoxide substrate (14). 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). Currently we are trying to understand the scope of the reactions catalyzed by this catalase-related family of enzymes. Our objectives are to help understand the structure-function relationships in the reactions with peroxides, to provide new insights on the mechanism of these hemoprotein-catalyzed transformations, as well as, by reflection, to give a different perspective on the parent hemoprotein, the hydrogen peroxide-metabolizing true catalase.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). Reaction is generally considered to be initiated by homolytic cleavage of the peroxide O–O bond (see Fig. 1). While subsequent reactions can be construed as following a radical pathway to product (e.g. in the hydroperoxide lyase reaction (19)), others are considered to involve an electron transfer step, giving a carbocation intermediate in the penultimate steps to product (22) (Fig. 1). The underlying grounds for these mechanisms are more a subject of debate and of comparison to other chemistry than of defining evidence on the reactions in question. The results reported here add a measure of experimental support for ionic events in the peroxide transformation by a catalase-related hemoprotein.Open in a separate windowFIGURE 1.Hemoprotein-catalyzed transformation of fatty acid hydroperoxides via diverging routes. A radical pathway can lead to aldehydes (left side) and an ionic pathway via a putative carbocation intermediate to allene oxides (right side).Recently genome sequencing was completed on the cyanobacterium Acaryochloris marina (23). A. marina is a focus of attention owing to its harboring a light-harvesting complex containing the unusual chlorophyll d, considered a bridge in the evolutionary development of photosynthetic mechanisms (24, 25). BLAST searches of the A. marina genome reveal three individual LOX sequences and additionally, 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 (C18.2ω6); the partial incorporation of ¹⁸O from water in a newly formed hydroxyl group has implications related to the mechanism of hydroperoxide transformation.