Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase
Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase
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DOI：
10.1128/jb.01001-13
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发表时间：
2013-12-01
影响因子：
3.2
通讯作者：
Maier, Robert J.
中科院分区：
文献类型：
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作者：
Benoit, Stephane L.;Bayyareddy, Krishnareddy;Maier, Robert J.
蛋白质暴露于 HOCl 等氧化剂会导致蛋氨酸亚砜 (MetSO) 残留物的形成，该残留物可被蛋氨酸亚砜还原酶 (Msr) 修复。幽门螺杆菌 msr 菌株对次氯酸介导的杀灭作用比亲本更敏感。由于幽门螺杆菌中含量丰富且蛋氨酸含量高，烷基氢过氧化物还原酶 C (AhpC) 被假设易于发生蛋氨酸氧化。 AhpC 在大肠杆菌中表达为重组蛋白。 AhpC 活性被 HOCl 消除，而该酶的所有六个蛋氨酸残基都被完全或部分氧化。与 Msr 修复混合物一起孵育后，AhpC 活性恢复至非氧化水平，并且 MetSO 残基修复为蛋氨酸，尽管程度不同。 AhpC 中两个氧化程度最高且随后被 Msr 修复的蛋氨酸残基 Met101 和 Met133 通过定点诱变单独或一起被异亮氨酸残基取代。表达变体版本的大肠杆菌细胞比表达天然蛋白的细胞对叔丁基氢过氧化物更敏感，并且纯化的 AhpC 变体蛋白具有天然酶活性的 5% 至 39%。变体蛋白仍然能够像天然版本一样寡聚，并且变体蛋白的圆二色性 (CD) 光谱显示 AhpC 构象没有显着变化，表明这些变体中活性的丧失与主要结构改变无关。我们的结果表明，Met101 和 Met133 残基对于 AhpC 催化活性都很重要，并且它们的完整性依赖于功能性 Msr 的存在。
Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met101 and Met133, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met101 and Met133 residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.