Essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: Genetic and kinetic characterization

Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely relat ed to 2-Cys peroxiredoxins of higher organisms than to most other eubacteri al AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essen tial, suggesting a critical role for AhpC in defending H. pylori against ox ygen toxicity. Characterization of the ahpC promoter region divulged two pu tative regulatory elements and identified the transcription initiation site , which was mapped to 96 and 94 bp upstream of the initiation codon, No hom ologue of ahpF, which encodes the dedicated AhpC reductase in most eubacter ia, was found in the H, pylori genome. Instead, homologues of Escherichia c oli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) forme d a reductase system for H,pylori AhpC, A second Trx homologue (Trx2, HP145 8) was identified but was incapable of AhpC reduction, although Trx2 exhibi ted disulfide reductase activity with other substrates [insulin and 5,5'-di thiobis(2-nitrobenzoic acid)], AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V-max and K- m values) but rapid enough (at 1 x 10(5) to 2 x 10(5) M-1 s(-1)) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhi bited a Hide specificity for hydroperoxide substrates, which, taken togethe r with the above results, suggests a minimal binding site for hydroperoxide s composed of little more than the cysteinyl (Cys49) active site. H. pylori AhpC was not reduced by Salmonella typhimurium AhpF and was slightly more active with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrati ng the specialized catalytic properties of this peroxiredoxin.