Isoniazid‐resistance conferring mutations inMycobacterium tuberculosisKatG: Catalase, peroxidase, and INH‐NADH adduct formation activities

Abstract

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD 1 /NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-TyrTrp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H 2 O 2 ), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct.

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

“…A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.…”

“…Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.…”

Virulence Factors and Pathogenicity of Mycobacterium

“…Although isoniazid resistance is multigenic, mutations in katG predominate among the INH-resistant strains, but the effect of these mutations on MTB virulence is variable. In general, katG mutations render MTB strains sensitive to endogenous or exogenous peroxides, generated during bacteria respiration or by phagocytes during infection [105], maybe because mutations also affect the peroxidase domain of KatG [106] though the activity of the alkyl hydroperoxidase C (AhpC) has been described as an important compensatory mechanism in INH-resistant strains [107].…”

Pyrimidine Incorporated Schiff Base of Isoniazid With Their Synthesis, Characterization and in Vitro Biological Evaluation

“…It is prodrug which is activated by Mycobacterium tuberculosis catalase-peroxidase (Kat G) is a bifunctional hemoprotein [1]. The primary target of isoniazid (INH) is the enoyl-ACP reductase enzyme (InhA) from M. tuberculosis.…”