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Abstract
Helicobacter pylori depends upon the Ni2+-dependent metalloenzyme urease for the catalytic hydrolysis of urea into ammonia and bicarbonate for colonization of the human stomach. The produced ammonia acts as a buffer against the low pH of the stomach lumen. After colonization in the gastric mucosa, H. pylori is able to persist in the stomach for the lifespan of the host. Urease is the fourth most abundant protein in H. pylori and has a relatively high percentage of methionine (Met) residues. Based on these unique characteristics of urease, as well as the activity of methionine sulfoxide reductase (Msr), it was proposed that urease could act in a noncatalytic manner to quench oxidants through a Met residue oxidation-reduction cycle. To test this hypothesis, I constructed site directed mutants that were catalytically inactive due to an inability to bind nickel, but they still produced urease as detected by western blot. These apo-urease mutants were significantly more resistant to hypochlorous acid (HOCl) than a complete urease deletion mutant (ureAB). Purified apo- and holo-urease were able to protect H. pylori against HOCl in both the wildtype and in a ureAB background. To further characterize the role Met residue recycling plays in oxidant quenching, a mass spectrometry (MS) approach was used. Unoxidized, oxidized, and oxidized and then Msr-repaired urease was analyzed by MS/MS to identify the Met residues susceptible to oxidation and repair. Of the 25 Met residues of urease, 11 were subject to both oxidation and Msr-mediated repair. This provides evidence for a noncatalytic antioxidant role for urease whereby oxidized urease Met residues are reduced to create a recyclable sink for oxidants. This new role for urease could contribute to the extended persistence of H. pylori infection. Additionally, I explored possible oxidant quenching in other Met-rich H. pylori proteins as well as targeting of Ni metalloenzymes (e.g. hydrogenase and urease) with a nickel chelator as a new antimicrobial mechanism.