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

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 18.43 and 18.33 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 ϳ70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only ϳ30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H 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), ␣-dioxygenase in plants (2), and several linoleate dioxygenases in fungi (3-5). The nonheme 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 A 4 synthase (9) or aldehyde-synthesizing hydroperoxide cleaving activity (10) among the LOX 2 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 18). Currently we are trying to understand the scope of the reactions catalyzed by this catala...

On the Role of Molecular Oxygen in Lipoxygenase Activation

“…And 15S-HPETE is converted via either C-7 or C-10 centered radicals to allylic epoxides such as 14,15-LTA 4 (Fig. 11B) (25). This O 2 -independent enzyme activation is best explained if a fraction of the alkoxyl/epoxyallylic radical intermediate is released into the solution, probably because of a low affinity of the enzyme for this radical intermediate, leaving the enzyme in the activated ferric state.…”

“…15S-HPETE or 13S-HPODE was synthesized by reacting soybean LOX-1 with arachidonic acid or linoleic acid at pH 9 followed by SP-HPLC purification (24). [ 18 O]13S-HPODE was synthesized under 18 O 2 atmosphere (25). Nordihydroguaiaretic acid (NDGA) was purchased from Cayman Chemical Co. (Ann Arbor, MI).…”

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

“…The tomato CYP74C3 plasmid was s-cis conformation 9 a gift of Dr. Gregg Howe (Michigan State University) and was expressed and purified as described (19). The guayule CYP74A2 in plasmid pET28b was a gift of Drs.…”

“…Based on the very similar appearance of the double bond protons in the spectra, this generality applies to the allene oxides formed by cytochrome P450 CYP74A (10), by the catalase-related AOS in coral (substrate 8R-hydroperoxyeicosatetraenoic acid) (11,29), and by the catalase-related AOS of the cyanobacterium Acaryochloris marina (substrate 12R-hydroperoxy linolenic acid) (9) (Fig. 7).…”

“…In coral, the lipoxygenase precursor is the 8R-hydroperoxide of arachidonic acid, which is transformed by a catalase-related hemoprotein to the 8,9-epoxy allene oxide, a presumed precursor of the clavulones and related cyclopentenones of corals (7,8). Recently, an allene oxide formed from 12R-hydroperoxy linolenic acid by a cyanobacterial catalase-related enzyme was described (9).…”