Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori : Genetic and Kinetic Characterization

Abstract

Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H. pylori against oxygen toxicity. Characterization of the ahpC promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of ahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H. pylori genome. Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for H. pylori AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5-dithiobis(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 ؋ 10 5 to 2 ؋ 10 5 M ؊1 s ؊1 ) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides 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, demonstrating the specialized catalytic properties of this peroxiredoxin.

Insights on the Evolution of Metabolic Networks of Unicellular Translationally Biased Organisms from Transcriptomic Data and Sequence Analysis

“…Our analysis also indicates the thioredoxin pathway (40) to have a rather high RPI value (see Fig. 1), and this is experimentally confirmed by the finding that H. pylori has a thioredoxin-dependent peroxiredoxin system playing a critical role in the defense against oxygen toxicity that is essential for survival and growth, even in microaerophilic environments (Baker et al 2001). Another highly RPI ranked pathway is riboflavin biosynthesis (48), which turns out to play a crucial role in ferric iron reduction and iron acquisition by H. pylori (Worst et al 1998).…”

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

“…It should, of course, be emphasized that other important bacteria such as Pseudomonas aeruginosa have been demonstrated to contain millimolar levels of GSH (22); still they express only the type III PanK (19,20). Analysis of the Helicobacter pylori genome has been reported to reveal the absence of gshA, gor, and grx genes (66), implying the lack of a GSH/GSSG/glutathione reductase thiol/disulfide redox system. Thus, of the five known functional type III PanKs, only one is derived from a GSH-dependent microorganism (P. aeruginosa).…”

The Peroxiredoxin Repair Proteins

“…The YF helix and GGLG structural motifs, as described earlier, promote hyperoxidation and have been identified in most eukaryotic organisms and cyanbacteria . Many bacteria contain a GG(L/I)G motif, but the critical YF-motif is substituted with a YL motif resulting in a Prx that is insensitive to hyperoxidation (Baker et al, 2001). Interestingly, these bacteria do not contain the genes for sulfiredoxin or sestrin.…”