Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

Abstract

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353-356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347-357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a "biomimetic" chemical method originally described for LTA(4) synthesis and showed that like LTA(4), the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.

Fig. 1.

RP-HPLC analysis of the stable end products of linoleic acid metabolism by the Anabaena peroxidase-LOX fusion protein. Column: Waters Symmetry C18, 25 × 0.46 cm; solvent, methanol/water/glacial acetic acid (65:35:0.01, by volume); flow rate, 1 ml/min; UV detection at 235 nm. Inset: the UV spectra of peaks 1–4; the spectra of peaks 1 and 2 are identical to each other (λmax 230.5 nm), while 3 is shifted to 231.5 nm and 4 (dashed line) to 231.8 nm.

Fig. 2.

Chromatography of the linoleate allylic epoxide. A: The sample was run on an Altex/Beckman silica column (5 μ, 4.5 × 0.46 cm) immersed in an ethanol/ice bath at approximately −10 to −15°C with a solvent of hexane/diethyl ether (100:5, v/v) at a flow rate of 3 ml/min with UV detection at 235 nm. B: UV spectrum of the linoleate allylic 9,10-epoxide prepared using the Anabaena enzyme. For comparison, the spectrum of 9-HPODE is shown as the dotted line. Solvent: hexane.

Fig. 3.

¹H-NMR spectrum of the methyl ester of the unstable epoxide formed from linoleic acid by the Anabaena peroxidase-LOX fusion protein. The inset displays the olefinic protons in more detail. The product is identified as 9R,10R-epoxyoctadeca-11E,13E-dienoate methyl ester. Solvent: d6-benzene. Chemical shift δ, multiplicity, number of protons, proton number, J: 6.38, dd, 1, H12, J_11,12 = 15.6, J_12,13 = 10.6; 6.04, dd, 1, H13, J_12,13 = 10.7, J_13,14 = 15.0; 5.59, td, 1, H14, J_13,14 = 15.1, J_14,15 = 7.1; 5.35, dd, 1, H11, J_10,11 = 8.0, J_11,12 = 15.4; 3.36, s, 3, CH₃O; 3.02, dd, 1, H10, J_9,10 = 1.3, J_10,11 = 8.0; 2.69, dt, 1, H9, J_8,9 = 6.3, J_9,10 = 1.7; 2.10, t, 2, H2; 1.95, q, 2, H15; 1.53, p, 2, H3; 1.45–1.33, m, 2, H8; 1.28–1.26, m, 4, H16,17; 1.15–1.06, m, 8, H4,5,6,7; 0.86, t, 3, H18.

Fig. 4.

UV spectra of the 12,13-epoxides and a glutathione conjugate. A: The two epoxides formed by chemical synthesis from 13-HPODE methyl ester, recorded in hexane. B: The GSH conjugate of epoxide 2, the 12,13-epoxy-octadeca-8E,10E-dienoate recorded in methanol. The spectrum of 13-HPODE is included as the dotted line in the figures for comparison.

Fig. 5.

Mechanisms of transformation to allylic epoxides. A: LOX-catalyzed LTA-type epoxide biosynthesis. B: Transformation of 9R-HPODE by the Anabaena enzyme. C: Chemical biomimetic synthesis of allylic epoxide.