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, Met 101 and Met 133 , 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 Met 101 and Met 133 residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.

Helicobacter Catalase Devoid of Catalytic Activity Protects the Bacterium against Oxidative Stress

“…These results suggest that catalase, even when inactive and lacking the heme moiety, enables H. pylori cells to combat HOCl. In addition, ⌬msr single mutants were significantly (p Ͻ 0.01) more sensitive to HOCl than the WT strain, in agreement with previously published results (26). Double mutants ⌬katA ⌬msr, katA H56A ⌬msr, and katA Y339A ⌬msr were also more sensitive than WT (p Ͻ 0.01).…”

“…Then 0.9-ml aliquots of this suspension were incubated for 60 min at 37°C (no shaking) with either 0.1 ml of PBS, 0.1 ml of PBS with NaOCl (10 -15% available chlorine; Sigma), or 0.1 ml of PBS/NaOCl that had been previously incubated for 15 min at 37°C in the presence of purified protein Kat WT , Kat H56A , Kat Y339A , HypC, or UreE. Both HypC and UreE were expressed and purified as described previously (26,36). Final protein and NaOCl concentrations were 0.25 and 200 M, respectively, and final A 600 was equal to 1 in each tube (ϳ5 ϫ 10 8 cells per ml).…”

Protein Quality Control under Oxidative Stress Conditions

“…In humans, MSR-catalyzed methionine sulfoxide reduction restores chaperone function to oxidatively inactivated a-crystallin (55). In the bacterial pathogen Helicobacter pylori , resistance to oxidative stress depends on MSR not only for repairing oxidized methionines in the ROS-degrading enzymes catalase and alkyl hydroperoxide reductase, but also for restoring activity to oxidatively-inactivated GroEL chaperones (56–58). Like cysteine, under severe oxidizing conditions methionine can also be further oxidized to methionine sulfone or methionine sulfoximine.…”

Methionine sulfoxide reductase A (MsrA) contributes to Salmonella Typhimurium survival against oxidative attack of neutrophils

“…HOCl. msr gene deletion strains of E. coli [27] and H. pylori [28] were showed hypersensitivity to HOCl. In E. coli hypersensitivity to HOCl was found to be due to Met oxidation as the quantum of protein Met-SO formation and E. coli killing were directly dependent to HOCl dose.…”