Abstract
The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF from Salmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H(2)O(2) but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies.
Highlights
Tuberculosis, caused by opportunistic infection by Mycobacterium tuberculosis, is a leading cause of death [1]
Middlebrook and coworkers [4] observed in the 1950s that M. tuberculosis strains resistant to isoniazid were devoid of catalase/peroxidase activity. This circumstantial link between peroxidase and isoniazid activities was placed on a molecular footing by Heym et al [5, 6], who showed that isoniazid-resistant M. tuberculosis strains had deletions or mutations in the katG gene that encodes for the KatG catalase/peroxidase
Analysis of the genes induced in isoniazid-resistant M. tuberculosis indicates that one of the mechanisms used by the organism to compensate for loss of the KatG antioxidant activity is to up-regulate the ahpC gene product, which codes for a non-hemoprotein alkylhydroperoxidase [15,16,17,18,19]
Summary
Johnson and Schultz [8] showed that isoniazid is oxidized by the M. tuberculosis KatG catalase/peroxidase to a number of chemically reactive products These combined results imply that isoniazid is a prodrug that must be processed into its active form by the bacterial cell. Analysis of the genes induced in isoniazid-resistant M. tuberculosis indicates that one of the mechanisms used by the organism to compensate for loss of the KatG antioxidant activity is to up-regulate the ahpC gene product, which codes for a non-hemoprotein alkylhydroperoxidase [15,16,17,18,19]. The most studied member of the alkylhydroperoxidase family is the enzyme from Salmonella typhimurium [20, 21] This protein contains two cysteine sulfhydryls that catalyze the reduction of peroxides to the corresponding alcohols and water with concomitant oxidation of the cysteine residues to give a disulfide bond (Scheme 1). We report here the first expression and purification of the M. tuberculosis AhpC and AhpD proteins and their initial structural and catalytic characterization
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