Formation of a cyclopropyl epoxide via a leukotriene A synthase-related pathway in an anaerobic reaction of soybean lipoxygenase-1 with 15S-hydroperoxyeicosatetraenoic acid: evidence that oxygen access is a determinant of secondary reactions with fatty acid hydroperoxides

Abstract

The further conversion of an arachidonic acid hydroperoxide to a leukotriene A (LTA) type epoxide by specific lipoxygenase (LOX) enzymes constitutes a key step in inflammatory mediator biosynthesis. Whereas mammalian 5-LOX transforms its primary product (5S-hydroperoxyeicosatetraenoic acid; 5S-HPETE) almost exclusively to LTA(4), the model enzyme, soybean LOX-1, normally produces no detectable leukotrienes and instead further oxygenates its primary product 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mammalian 15-LOX-1 displays both types of activity. We reasoned that availability of molecular oxygen within the LOX active site favors oxygenation, whereas lack of O(2) promotes LTA epoxide synthesis. To test this, we reacted 15S-HPETE with soybean LOX-1 under anaerobic conditions and identified the products by high pressure liquid chromatography, UV, mass spectrometry, and NMR. Among the products, we identified a pair of 8,15-dihydroxy diastereomers with all-trans-conjugated trienes that incorporated (18)O from H(2)(18)O at C-8, indicative of the formation of 14,15-LTA(4). A pair of 5,15-dihydroxy diastereomers containing two trans,trans-conjugated dienes (6E,8E,11E,13E) and that incorporated (18)O from H(2)(18)O at C-5 was deduced to arise from hydrolysis of a novel epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-cyclopropyl]-eicosa-5Z,7E,13E-trienoic acid. Also identified was the delta-lactone of the 5,15-diol, a derivative that exhibited no (18)O incorporation due to its formation by intramolecular reaction of the carboxyl anion with the proposed epoxide intermediate. Our results support a model in which access to molecular oxygen within the active site directs the outcome from competing pathways in the secondary reactions of lipoxygenases.

FIGURE 1.

RP-HPLC analysis of products from the anaerobic reaction of soybean LOX-1 (0.38 μm) with 15S-HPETE (40 μm). The products were eluted using a Waters Symmetry C₁₈ column (0.46 × 25 cm), a solvent system of methanol/water/acetic acid (80:20:0.01 by volume), and a flow rate of 1 ml/min. Shown are the UV traces at 205, 235, and 270 nm monitored by an Agilent diode array detector. The products are designated 1–7 based on the order of elution.

FIGURE 2.

RP-HPLC analysis of products from the anaerobic reaction of soybean LOX-1 (0.38 μm) with 15S-HPETE (40 μm) in the presence of NDGA (80 μm). As in Fig. 1, the products were eluted using a Waters Symmetry C₁₈ column (0.46 × 25 cm), a solvent system of methanol/water/acetic acid (80:20:0.01 by volume), and a flow rate of 1 ml/min. Shown are the UV traces at 205, 235, and 270 nm monitored by an Agilent diode array detector.

SCHEME 1.

Structures of products 1, 6, and 7.

FIGURE 3.

SP-HPLC analysis of products from the anaerobic reaction of soybean LOX-1 (0.38 μm) with [1-¹⁴C]15S-HPETE (40 μm) in the presence of 13S-HPODE (80 μm). A, the ¹⁴C radiochromatogram recorded by a radioactive detector. B, the UV chromatogram at 235 and 270 nm recorded by a diode array detector. The products were eluted using a Beckman Ultrasphere® silica column (0.46 × 25 cm), a solvent system of hexane/isopropyl alcohol/acetic acid (100:5:0.1 by volume), and a flow rate of 1 ml/min.

FIGURE 4.

Comparison of the UV spectra of products 4 and 5 with that of 5,15-(E,Z,Z,E)-diHETE. A, the spectra in RP-HPLC solvent, methanol/water/acetic acid (80:20:0.01 by volume). B, the spectra in SP-HPLC solvent, hexane/isopropyl alcohol/acetic acid (100:5:0.1 by volume).

FIGURE 5.

¹H NMR analysis of products 4 (A) and 5 (B) in d₆-benzene. Shown is an expanded view of the region corresponding to olefinic protons. The splitting pattern of each olefinic proton is also illustrated. The proton signals are assigned with aid of ¹H,¹H COSY NMR (supplemental Figs. S6 and S8).

FIGURE 6.

GC-MS (electron ionization) analysis of TMS-derivatized hydrogenated products 4 and 5 from the anaerobic reaction in H₂¹⁸O. A, the mass spectrum of the TMS ether TMS ester derivative of hydrogenated product 4. B, the mass spectrum of the TMS ether derivative of hydrogenated product 5.

FIGURE 7.

The proposed mechanism of formation of products 4 and 5.

FIGURE 8.

Comparison of the aerobic and anaerobic reactions of soybean LOX-1 with 15S-HPETE. Aerobic reaction is initiated by the H-7 and H-10 hydrogen abstractions (pro-R hydrogens, designated as in arachidonic acid rather than 15S-HPETE) with 15S-HPETE in the reversed orientation, antarafacial oxygenation giving the 5S,15S-diHPETE and 8S,15S-diHPETE products, respectively (6, 44, 45). Anerobically, analogous hydrogen abstractions can account for formation of the cyclopropyl epoxide and 14,15-LTA₄, respectively, followed by non-enzymatic hydrolysis to give the stable diol end products.