Institute for Food Toxicology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany.
Institute of Biochemistry, University Medicine Berlin - Charité, Charitéplatz 1, 10117 Berlin, Germany.
Biochim Biophys Acta Mol Cell Biol Lipids. 2017 Jul;1862(7):666-675. doi: 10.1016/j.bbalip.2017.04.001. Epub 2017 Apr 8.
Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n-6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n-3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n-6 and n-9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n-3 and n-6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n-3 polyunsaturated fatty acids.
哺乳动物脂氧合酶（LOX）已被牵连到细胞分化以及炎症、过度增殖和神经病变等疾病的发病机制中。尽管已经详细研究了哺乳动物 LOX 与 n-6 脂肪酸（亚油酸、花生四烯酸）的反应特异性，但对于 n-3 多不饱和脂肪酸形成的产物模式的信息很少，这些脂肪酸在营养上尤为重要，并且是合成促分解炎症介质（如 resolvins 和maresins）的底物。在这里，我们表达了 8 种不同哺乳动物的 ALOX15 直系同源物以及人 ALOX12 和 ALOX15B 作为重组 His 标记融合蛋白，并对它们与最丰富的多不饱和脂肪酸（PUFA）的反应特异性进行了表征，包括 5,8,11,14,17-二十碳五烯酸（EPA）和 4,7,10,13,16,19-二十二碳六烯酸（DHA）。我们发现，所测试的 LOX 同工型接受这些脂肪酸作为合适的底物，并以可变的位置特异性将它们氧化为相应的 n-6 和 n-9 氢过氧化物衍生物。令人惊讶的是，人 ALOX15 以及黑猩猩和猩猩的相应直系同源物，主要将花生四烯酸氧化为 15S-H(p)ETE，表现出明显的双重反应特异性，与 DHA 形成相似量的 14-和 17-H(p)DHA。此外，当提供等摩尔浓度的 n-3 和 n-6 PUFA 时，ALOX15 直系同源物优先选择 DHA 和 EPA 而不是 AA。总的来说，这些数据表明哺乳动物 LOX 同工型的反应特异性是可变的，并且强烈依赖于脂肪酸底物的化学性质。大多数哺乳动物 ALOX15 直系同源物对高度不饱和的 n-3 多不饱和脂肪酸具有双重位置特异性。
