Characterization of site-specific mutants of alkylhydroperoxide reductase with dual functionality from Helicobacter pylori

Alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in Helicobacter pylori (HP), a spiral Gram-negative microaerophilic bacterium. By sequence alignment and structure comparison, HP-AhpC was found to be more homologous to human peroxiredoxins (hPrx) than to other eubacterial AhpC proteins. Similar to hPrxI, native HP-AhpC existed as a dimer of single subunit, comprising alpha-helix and beta-sheet domains with low surface hydrophobicity. AhpC can form high-molecular-weight (HMW) aggregates ranging from 700 to higher than 2,000 kDa under oxidative stress, possessing chaperone activity in the presence of thioredoxin (Trx). Further analysis of peroxide-reductase activities showed that HP-AhpC was more resistant to H(2)O(2) than hPrxI. However, the mechanism of enzyme inactivation to H(2)O(2) appeared to be similar for both HP-AhpC and hPrxI as revealed by native gel electrophoresis followed by proteomic identification using two-dimensional gel electrophoresis (2-DE) and LC-MS/MS. In contrast to T90D-hPrxI mutant with chaperone activity, site-specific mutant T87D-HP-AhpC did not form HMW chaperone complexes. The comparison of these two evolutionarily distant and yet functionally related enzymes may shed some light on the mechanism(s) underlying the evolution and development of the dual functionality in HP-AhpC and hPrxI with similar protein structure.