Efficient and Specific Conversion of 9‐Lipoxygenase Hydroperoxides in the Beetroot. Formation of Pinellic Acid

Abstract

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.

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

“…The majority of these are mechanistically quite distinct, however, because the epoxide is formed via oxygen transfer. The epoxyalcohol synthase activities in the fi sh parasitic fungus Saprolegnia parasitica ( 42 ) and in potato leaves and beetroot ( 43,44 ) catalyze oxygen transfer from the hydroperoxy fatty acid to the adjacent conjugated diene; the original hydroperoxide moiety is reduced to an alcohol, while the transferred oxygen produces trans -or cis -epoxidation of the trans and cis double bonds, respectively. In the case of plant peroxygenases, epoxidation may occur via intermolecular or intramolecular oxygen transfer from a fatty acid hydroperoxide to a cis double bond ( 45,46 ).…”

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

“…Heme and lipoxygenase catalysis involves formation of alkoxyl and epoxyallylic radical intermediates, whereas oxygen transfer from the hydroperoxide with epoxidation of a remote double bond is effected by other enzymes. These include peroxygenase (Blée et al, 1993; Hamberg and Hamberg, 1996), catalase-related hemoproteins (Gao et al, 2009; Niisuke et al, 2009), as well as putative P450-like enzymes that remain to be characterized, for example in the fish fungus Saprolegnia parasitica (Hamberg et al, 1986), potato leaves (Hamberg, 1999), and beetroot (Hamberg and Olsson, 2011). …”

“…iii) To utilize a GC-MS and NMR approach for the assignment of the trihydroxy-octadecenoate regio- and stereo-chemistry. iv) To compare the enzymatic synthesis, isomeric distribution and chirality of epoxyalcohols and triols from 9 R - and 9 S -hydroperoxy-linoleic acid in Beta vulgaris , a source of epoxyalcohol synthase (Hamberg and Olsson, 2011), and v) To characterize specific delta-ketols as products from the mild acid treatment of allylic trans -epoxyalcohols.…”

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

“…The former mechanism has been observed in the vanadium-promoted degradation of hydroperoxides (3), for peroxygenase (4,5) and for a number of unidentified epoxy alcohol synthases present in certain plants and fungi (ref. 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).…”