8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

Abstract

This article is available online at http://www.jlr.org dins for research ( 3 ). Due to differences in the prostaglandin profi le from that typically seen in mammalian systems, for some time it was suspected that there existed a different prostaglandin biosynthetic pathway in coral ( 4,5 ). Although this putative noncyclooxygenase pathway of prostaglandin synthesis turned out to be a red herring and cyclooxygenase accounts for the biosynthesis ( 6, 7 ), research into polyunsaturated fatty acid metabolism in coral extracts uncovered other interesting biochemistry. Bundy and colleagues studied the related coral Pseudoplexaura porosa and uncovered 8 R -lipoxygenase activity, the fi rst known existence of an R -specifi c lipoxygenase ( 8 ). 8 R -LOX was subsequently found to be widespread in corals including in P. homomalla ( 5, 9 ) as well as in many marine invertebrates ( 10 ), and a 12 R -LOX is highly conserved and functionally essential in mammals ( 11,12 ). A second novel activity detected in coral extracts was allene oxide synthase ( 5, 13 ), which transforms the 8 R -LOX product, 8 R -hydroperoxy-eicosatetraenoic acid, to an allene epoxide ( 5 ), a proposed intermediate in biosynthesis of cyclopentenones such as the clavulones ( 14-16 ), and which hydrolyzes in vitro to an ␣ -ketol derivative ( 5 ).The work described in the present paper was initiated in the early 1990s, before the cloning of P. homomalla cyclooxygenases and lipoxygenases. It concerns an unexpected difference we observed in the metabolism of arachidonic acid (20:4 6) and dihomo-␥ -linolenic acid (20:3 6) in extracts of P. homomalla ; a prominent, relatively polar, product is formed specifi cally from 20:3 6. This difference had been noted before in work from the E. J. Corey laboratory ( 17 ).Abstract Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S -specifi c lipoxygenases forming epoxyalcohols with a trans -epoxide confi guration. Here we report on R -specifi c lipoxygenase synthesis of a cis -epoxyalcohol. Although arachidonic and dihomo-␥ -linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8 R -lipoxygenase and allene oxide synthase activities, 20:3 6 forms an additional prominent product, identifi ed using UV, GC-MS, and NMR in comparison to synthetic standards as 8 R ,9 S -cis -epoxy-10 S -erythro -hydroxy-eicosa-11 Z ,14 Zdienoic acid. Both oxygens of In the late 1960s and early 1970s, high concentrations of prostaglandin esters were identifi ed in the Caribbean sea whip coral Plexaura homomalla ( 1, 2 ), and for a few years this abundant octacoral served as a source of prostaglan-

WITHDRAWN: Structural and mechanistic studies of hydroperoxide conversions catalyzed by a CYP74 clan epoxy alcohol synthase from amphioxus (Branchiostoma floridae)

“…The double bond has the "Z" configuration as seen from the J 9,10 = 11 Hz, and the epoxide is cis-configured as shown by the J 12,13 = 4.3 Hz. The spin-spin interaction between H11 and H12 (J 11,12 = 7.7 Hz) does not allow assignment of the erythro/threo configuration because of the small differences between the relevant coupling constants in the NMR spectra of erythro and threo cis-epoxyalcohols (12). However, the 2D-NOESY spectrum (data not shown) revealed an intense cross-peak between H11 and H12 as well as a less prominent cross-peak between H11 and H13, and these nuclear Overhauser effects indicate the spatial proximity between these protons (especially H11 and H12) in agreement with the threo configuration.…”

“…6, and references cited therein). The latter mechanism most often results in the formation of trans-configured epoxy alcohols and has been observed for various nonenzymatic (7,8) and lipoxygenase-promoted conversions (9,10), as well as for specific enzymatic hydroperoxide isomerizations catalyzed by lipoxygenase-type enzymes (11,12). 0.5 M urea.…”

Secretion of two novel enzymes, manganese 9S-lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii

“…Epoxy alcohols can be formed by ferrous and ferric hemoproteins, designated <Fe 2+ > and <Fe 3+ >, respectively, by certain LOXs and by EASs. The scheme shows within brackets to the right, heterolytic cleavage of the O-O bond, probably with formation of a hydroxyl anion and ferryl oxygen (not shown) in analogy with peroxidases ( 49 ), and to the left, homolytic cleavage with formation of a trans epoxide and a dislocated radical at C-11 to C-13 [or, in at least one case, formation of a cis epoxide and the disclocated radical ( 42 )]. The latter are formed from a peroxyl radical at C-9 (not shown).…”

“…7C ). The rate constants for of LOXs and ferrous hemoproteins ( 42,49 ). A schematic overview of the mechanism of biosynthesis of epoxy alcohols by metal enzymes is summarized in Fig.…”

“…EAS transformed 9 S -HPODE to an epoxy alcohol. It is well known that isomers of 9(10)-epoxy-11-hydroxy-12 -Z -18:1 can be formed from 9 S -HPODE by LOXs ( 41,42 ). During NP-HPLC, however, the EAS metabolite eluted between the erythro and threo isomers of 9(10)-epoxy-11-hydroxy-12 Z -18:1, which were obtained by hematin-catalyzed transformation of 9 S -HPODE ( Fig.…”