Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate

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Abstract
The pure reticulocyte lipoxygenase converts 15Ls‐hydroxy‐5,8,11,13(Z,Z,Z,E)‐icosatetraenoic acid (15Ls‐HETE) methylester to a complex mixture of products containing 5Ds,14LR,15Ls‐trihydro(pero)xy‐6E,8Z,10E,12E‐icosatetraenoate methyl ester (lipoxin B methyl ester), 5Ds,15Ls‐DiH(P)ETE methyl ester and four 8,15Ls‐DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy‐icosatetraenoic acid]. After a short incubation period (15 min) 5Ds,15Ls‐DiH(P)ETE methyl ester was found to be the main product, whereas after a 3‐h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400‐MHz 1H‐NMR. Atmospheric oxygen was incorporated at carbon‐5 and carbon‐14 into the major product. 5Ds,15Ls‐DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15Ls‐HETE and 5Ds,15Ls‐DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.