The extreme sensitivity of Mycobacterium tuberculosis to the front-line antituberculosis drug isoniazid

(6) further demonstrated that the inhibition of mycolic acid biosynthesis mediated by INH leads to the accumulation of a saturated C 26 fatty acid within 1 h of INH treatment. By using scanning electron microscopy, it was established that M. tuberculosis treated with INH undergoes a defined order of molecular events that leads to lysis 24 h after initiation of treatment, thus after approximately one cell generation (7). The events that lead from the inhibition of mycolic acid biosynthesis and the accumulation of a saturated C 26 fatty acid to cell lysis are still poorly understood. Moreover, there exists controversy as to which enzymes of the mycolic acid biosynthetic pathway are the actual targets of INH.The mode of action of INH appears to be rather complex and requires activation by the mycobacterial catalase-peroxidase KatG (8,9). Activation of the pro-drug leads to reactive radicals that exert a toxic effect on the tubercle bacillus (9 -11). Presumably activated INH inhibits one or more targets of the mycolic acid biosynthetic pathway which eventually leads to cell death. At least two enzymes have been identified as potential targets of activated INH, the enoyl-ACP reductase InhA (12) and the ␤-ketoacyl-ACP synthase KasA (13). The inhA gene was first identified by conferring co-resistance to INH and ethionamide (ETH) after overexpression in mycobacteria (12). In addition, mutations in inhA confer resistance to both INH and ETH in drug-resistant M. tuberculosis isolates (14 -17).

Section: Discussionmentioning

confidence: 99%

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

“…With NAD ϩ included in the mix, the generation of the isonicotinoyl-NAD adduct was observed both with and without KatG present (7)(8)(9), leading to the suggestions that the role of KatG is limited to the hydrazinolysis of INH and that the subsequent reaction of the isonicotinoyl radical with NAD ϩ is a nonenzymatic event involving a homolytic aromatic substitution (7,10). Reactive oxygen species have been implicated in INH activation both in vivo (11,12) and in vitro (6), and an elevated level of superoxide (13) was identified as a possible reason for the high sensitivity of M. tuberculosis to INH (1). However, the absence of H 2 O 2 involvement in INH activation implies that a reaction different from either the peroxidase or the catalase reactions is involved, and some reports have suggested that the active participation of KatG in INH activation involves more than just hydrazinolysis (9).…”

Section: Isonicotinic Acid Hydrazide (Isoniazid or Inh)mentioning

confidence: 99%

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Singh

Wiseman

Deemagarn

et al. 2004

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD ؉ and either H 2 O 2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (K m ‫؍‬ 12 M), but the oxidase reaction is slow (k cat ‫؍‬ 0.54 min ؊1 ) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen-and H 2 O 2 -independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD ؉ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD ؉ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD ؉ and INH more efficiently than B. pseudomallei KatG.

“…This environment includes peroxides formed by the oxidative burst, species such as peroxynitrite formed by the inducible nitric oxide synthase, and the alkyl peroxides that result from exposure of unsaturated lipids to oxidative stress. 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). Incubation of M. tuberculosis expressing elevated levels of the alkylhydroperoxidase with isoniazid has shown that the drug is not activated by this enzyme (15).…”

mentioning

confidence: 99%

The AhpC and AhpD Antioxidant Defense System of Mycobacterium tuberculosis

Hillas

Alba²,

Oyarzábal³

et al. 2000

130

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.