Department of Microbiology.
J Bacteriol. 2013 Dec;195(23):5396-401. doi: 10.1128/JB.01001-13. Epub 2013 Oct 4.
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.
蛋白质暴露于氧化剂（如 HOCl）会导致蛋氨酸残基氧化为亚砜（MetSO），这种氧化可以被蛋氨酸亚砜还原酶（Msr）修复。与亲本相比，幽门螺杆菌 msr 缺失菌株对 HOCl 介导的杀伤更为敏感。由于其在幽门螺杆菌中的丰度及其高蛋氨酸含量，烷基氢过氧化物还原酶 C（AhpC）被认为容易发生蛋氨酸氧化。AhpC 作为重组蛋白在大肠杆菌中表达。HOCl 使 AhpC 失活，而酶的六个蛋氨酸残基全部或部分被氧化。与 Msr 修复混合物孵育后，AhpC 活性恢复到非氧化水平，MetSO 残基被修复为蛋氨酸，尽管程度不同。AhpC 中两个最易氧化且随后被 Msr 修复的蛋氨酸残基，Met101 和 Met133，通过定点突变分别或一起被异亮氨酸残基取代。表达变异体的大肠杆菌细胞比表达天然蛋白的细胞对叔丁基过氧化氢更敏感，并且纯化的 AhpC 变异蛋白的活性为天然酶的 5%至 39%。变异蛋白仍然能够像天然版本一样聚合，并且变异蛋白的圆二色性（CD）光谱显示 AhpC 构象没有明显变化，表明这些变异体活性丧失与主要结构改变无关。我们的结果表明，Met101 和 Met133 残基对 AhpC 催化活性都很重要，其完整性依赖于功能性 Msr 的存在。
