Alkyl hydroperoxide reductase repair by Helicobacter pylori methionine sulfoxide reductase

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.

Fig 1

Sensitivity of nongrowing H. pylori wild-type and Δmsr mutant cells to HOCl. H. pylori SS1 (wild-type) and SS1 Δmsr mutant cells were grown on BA plates, harvested, and resuspended in PBS containing the indicated HOCl concentration for 30 min at room temperature. Cells were serially diluted and plated on BA plates, and CFU were counted after 3 to 5 days. Data shown represent the averages and standard deviations of the results of quadruplicate assays from one experiment, and the mutant is significantly more sensitive than the parent at 125 and 250 μM HOCl (P < 0.05, as determined by Student's t test). *, no CFU could be detected (detection limit, 100 CFU/ml).

Fig 2

Identification and quantification of HOCl-oxidized and Msr-repaired methionine residues in H. pylori AhpC by LC-MS/MS. AhpC was oxidized with a 60-fold molar excess of HOCl and subsequently repaired (or not) by Msr. Repaired and unrepaired AhpC samples were subjected to in-solution GluC digestion, and Met residues were analyzed and quantified by LC-MS/MS. The values corresponding to the percentages of oxidation (y axis) correspond to the ratios of MetO residues to the total number of times the peptide was found (Met coverage). Error bars represent 1 standard deviation (from three measurements). *, the unoxidized peptide was below our limit of detection.

Fig 3

SDS-PAGE of native and variant AhpC proteins. Oxidized native (wild-type) AhpC (WT_ox), nonoxidized native AhpC (WT), or variant AhpC (M₁₀₁I, M₁₃₃I) (2.5 μg per lane) proteins were mixed with reducing buffer and heated at 95°C for 5 min or mixed with nonreducing loading buffer (and not heated) before being separated on an SDS-12.5% polyacrylamide gel. A molecular mass standard (M) is shown on the right, with sizes indicated on the right. Calculated (theoretical) molecular mass of AhpC-His₆, 23.3 kDa.

Fig 4

CD spectra of purified native, M₁₀₁I variant, or M₁₃₃I variant AhpC proteins. CD spectra of each purified protein were measured using an Aviv 400 spectropolarimeter in a 0.1-cm-gap cuvette from 185 to 260 nm at 20°C. Mean residue molar ellipticities (θ) were calculated using a molar concentration of 6.4 μM and 206 amino acids.