Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes
Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes
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DOI：
10.1016/j.bbalip.2014.07.005
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发表时间：
2014-10-01
影响因子：
4.8
通讯作者：
Haefner, Ann-Kathrin
中科院分区：
文献类型：
--
作者：
Adel, Susan;Hofheinz, Katharina;Haefner, Ann-Kathrin
5-脂氧合酶（ALOX5）在促炎性白三烯的生物合成中起关键作用，而15-脂氧合酶（ALOX15）则与促溶解类二十烷酸（脂毒素，溶解蛋白）的形成有关。最近，有研究表明，人ALOX5稳定变体的磷酸化模拟突变体（Ser663Asp）表现出优势的花生四烯酸15-脂氧合酶活性（>95%）。为了检验是否在其他物种的ALOX5同源物中也能观察到类似的反应特异性改变，我们在原核和真核过表达系统中表达了人、小鼠和斑马鱼ALOX5的野生型和磷酸化模拟突变体（Ser271Asp、Ser523Asp、Ser663Asp、Ser663Glu），并表征了它们的反应特异性。我们发现两种磷酸化模拟突变体都没有产生大量的15-氢过氧二十碳四烯酸，所有野生型和突变型酶的5-脂氧合/15-脂氧合比率都低于100:2。综上所述，这些数据表明，不同脊椎动物的原生ALOX5同源物的磷酸化可能不会引起反应特异性的重大改变，因此可能不会逆转其生物活性。(C) 2014 Elsevier B.V.版权所有
5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (>95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity. (C) 2014 Elsevier B.V. All rights reserved.