Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase
Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase
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DOI：
10.1093/jb/mvm054
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发表时间：
2007-04-01
影响因子：
2.7
通讯作者：
Nishino, Takeshi
中科院分区：
文献类型：
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作者：
Asai, Ryosuke;Nishino, Tomoko;Nishino, Takeshi
活性氧是由NADPH氧化酶、黄嘌呤氧化还原酶（XOR）和线粒体呼吸酶等多种系统产生的，参与多种生理和病理过程。哺乳动物黄嘌呤脱氢酶（XDH）可以转化为黄嘌呤氧化酶（XO），其根据条件以约1：3的摩尔比产生超氧阴离子和过氧化氢。在这里，我们提出了一种突变的大鼠XOR，主要显示XO活性与超氧化物：过氧化氢的生产比例约为6：1。在突变体中，色氨酸335，这是从XDH到XO构象转换的关键氨基酸簇的组成部分，被丙氨酸取代，苯丙氨酸336，它通过与黄素辅因子的堆积相互作用调节FAD的氧化还原电位，被改变为亮氨酸。当突变体在Sf9细胞中表达时，它以XO形式获得，并且二硫苏糖醇处理仅部分恢复吡啶核苷酸结合能力。二硫苏糖醇处理的突变体在2.3埃分辨率下的晶体结构显示该酶的两个亚基非常相似，但不完全相同：参与构象转换的簇在一个亚基中完全被破坏，但在另一个亚基中仍然部分相关。该突变体中活性位点环的链迹与牛XO形式非常相似。这些结果与突变体的XDH和XO形式处于极大地有利于XO形式的平衡中的想法一致，但是在与二硫苏糖醇孵育后，平衡部分地向XDH形式移动。
Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 angstrom resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.