Adel Susan, Karst Felix, González-Lafont Àngels, Pekárová Mária, Saura Patricia, Masgrau Laura, Lluch José M, Stehling Sabine, Horn Thomas, Kuhn Hartmut, Heydeck Dagmar
Institute of Biochemistry, University Medicine Berlin-Charité, D-10117 Berlin, Germany;
Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain;
Proc Natl Acad Sci U S A. 2016 Jul 26;113(30):E4266-75. doi: 10.1073/pnas.1604029113. Epub 2016 Jul 13.
ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratLeu353Phe) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P < 0.01) [corrected]. Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory and proresolving lipoxins.
12/15-脂氧合酶（ALOX15）直系同源物与细胞内细胞器的成熟降解以及抗炎和促消退类二十烷酸的生物合成有关。在此，我们推测低等哺乳动物（小鼠、大鼠、猪）表达12-脂氧合的ALOX15直系同源物。相反，15-脂氧合亚型存在于高等灵长类动物（猩猩、人类）中，这些结果表明ALOX15特异性的进化。为了验证这一假设，我们首先克隆并鉴定了代表灵长类动物进化后期不同阶段的选定狭鼻猴类的ALOX15直系同源物，发现高等灵长类动物（人类、黑猩猩）表达15-脂氧合的直系同源物。相比之下，低等灵长类动物（狒狒、恒河猴）表达12-脂氧合酶。长臂猿在进化上介于恒河猴（12-脂氧合的ALOX15）和猩猩（15-脂氧合的ALOX15）之间，其表达的ALOX15直系同源物具有明显的双重特异性。为了探究这种进化改变的驱动力，我们对12-脂氧合（恒河猴、小鼠、大鼠、猪、人Ile418Ala）和15-脂氧合（人类、黑猩猩、猩猩、兔子、大鼠Leu353Phe）的ALOX15变体的脂氧素合酶活性进行了定量，发现当以花生四烯酸加氧酶活性进行标准化时，15-脂氧合的ALOX15变体的脂氧素合酶活性高出五倍多（P < 0.01）[校正后]。比较分子动力学模拟和量子力学/分子力学计算表明，对于15-脂氧合的兔子ALOX15，C13-氢提取（15-脂氧合）的能垒比花生四烯酸12-脂氧合的能垒低17 kJ/mol。相反，对于12-脂氧合的Ile418Ala突变体，15-脂氧合的能垒比12-脂氧合的能垒高10 kJ/mol。综上所述，我们的数据表明ALOX15特异性的进化旨在优化抗炎和促消退脂氧素的生物合成能力。
