Alkyl hydroperoxide reductase repair by Helicobacter pylori methionine sulfoxide reductase
Stéphane L Benoit1, Krishnareddy Bayyareddy, Manish Mahawar
1Department of Microbiology.
Abstract:
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.
Insights
Oxidative stress damages proteins, forming methionine sulfoxide (MetSO). Methionine sulfoxide reductase (Msr) repairs this damage. This study shows specific methionine residues in Helicobacter pylori alkyl hydroperoxide reductase C (AhpC) are crucial for its function and repair.
Area of Science:
- Biochemistry
- Oxidative Stress Biology
- Microbial Pathogenesis
Background:
- Oxidative stress, particularly from hypochlorous acid (HOCl), leads to methionine oxidation, forming methionine sulfoxide (MetSO) residues in proteins.
- Methionine sulfoxide reductases (Msr) are enzymes that repair MetSO residues, maintaining protein function.
- Helicobacter pylori, a pathogen, possesses abundant alkyl hydroperoxide reductase C (AhpC) with high methionine content, suggesting its susceptibility to oxidative damage.
Purpose of the Study:
- To investigate the susceptibility of H. pylori AhpC to oxidation by HOCl.
- To determine if Msr can repair oxidized AhpC and restore its activity.
- To identify specific methionine residues critical for AhpC catalytic activity and their reliance on Msr repair.
Main Methods:
- Recombinant expression of H. pylori AhpC in E. coli.
- Oxidation of purified AhpC using HOCl and subsequent repair using a Msr mixture.
- Site-directed mutagenesis to replace key methionine residues (Met101, Met133) with isoleucine.
- Enzyme activity assays and circular dichroism (CD) spectroscopy to assess protein function and structure.
Main Results:
- HOCl treatment abolished AhpC activity and oxidized its methionine residues.
- Msr-mediated repair restored AhpC activity and repaired MetSO residues to varying degrees.
- Mutant AhpC variants (Met101/133Ile) exhibited significantly reduced catalytic activity (5-39% of native).
- Structural integrity (oligomerization, CD spectra) was maintained in mutant variants, indicating specific residue importance over global conformational changes.
Conclusions:
- Met101 and Met133 residues are essential for the catalytic activity of H. pylori AhpC.
- The functional integrity of these critical methionine residues depends on a functional Msr repair system.
- AhpC's susceptibility to oxidation and subsequent repair highlights the importance of redox regulation in microbial enzyme function.
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