Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.
Abbreviations used: Ahp, alkylhydroperoxidase; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; GS, glutamine synthetase; MCO, metal-catalysed oxidation; Ni-NTA, Ni2+-nitrilotriacetate; Prx, peroxiredoxin.