Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C.
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.
Chauhan
Radha
R
Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India.
Mande
Shekhar C
SC
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004278:DNA, Superhelical; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics*; D054464:Peroxiredoxins; D010957:Plasmids / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004278:DNA, Superhelical; D004220:Disulfides; D029968:Escherichia coli Proteins; D013438:Sulfhydryl Compounds; D007501:Iron; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; D005974:Glutamate-Ammonia Ligase; D003545:Cysteine; D000409:Alanine; D010100:Oxygen; D004229:Dithiothreitol