Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential
Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential
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DOI：
10.1021/acs.biochem.5b01339
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发表时间：
2016-05-24
期刊：
影响因子：
2.9
通讯作者：
Holman, Theodore R.
中科院分区：
文献类型：
--
作者：
Green, Abigail R.;Barbour, Shannon;Holman, Theodore R.
脂氧素是一类重要的脂类介体，可通过花生四烯酸(AA)衍生的脂氧合酶产物的跨细胞交换来诱导炎症的消退。人上皮15-脂氧合酶-2(H15-LOX-2)是巨噬细胞中的主要脂氧合酶，具有严格的区域特异性，仅催化AA的碳15氢过氧化。为了确定H15-LOX-2在跨细胞合成事件中的催化潜力，我们将其与人类中三种脂氧合酶衍生的单氢过氧二十碳四烯酸(HPETE)反应：5-HPETE、12-HPETE和15-HPETE。H15-LOX-2仅以5-HPETE为底物，其稳态催化效率(k(CAT)/K-m)为AA的k(CAT)/K-m的31%。H15-LOX-2‘S与5-HPETE反应的唯一主要产物是脂氧素中间体5，15-二氢过氧二十碳四烯酸(5，15-diHPETE)。然而，H15-LOX-2不能与5，15-diHPETE进一步反应生成脂素。这一结果与H15-LOX-2的特异性一致，尽管5，15-diHPETE的反应性增加。密度泛函理论计算表明，从5，15-diHPETE中提取C10氢原子后，该自由基的能量比AA生成的相同自由基的能量低5.4kJ/mol，说明5，15-diHPETE可以生成脂素。有趣的是，H15-LOX-2确实与5S，6R-diHETE反应，生成Lipoxin A(4)，表明吉二醇不抑制H15-LOX-2的反应活性。综上所述，这些结果表明H15-LOX-2具有严格的区域特异性，限制了其在跨细胞合成中的作用。
Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (k(cat)/K-m) of this reaction was 31% of the k(cat)/K-m of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA(4), indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.