Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: 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(+)/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-Tyr-Trp 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.

Figure 1

Schematic representation of INH-NADH adduct formation as catalyzed by KatG via a putative isonicotinoyl radical.

Figure 2

Proposed reactions and putative intermediates of KatG involved in the oxidation of isoniazid.

Figure 3

Active site of Mtb KatG showing the heme prosthetic group, as well as the Met-Tyr-Trp crosslink. Coordinates (1SJ2) were obtained from the Protein Data Bank. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Crystal structure of the Mtb KatG dimer. The heme prosthetic group (red) and mutations examined in this study (Table I) are highlighted. Coordinates (PDB ID: 1SJ2) were obtained from the Protein Data Bank.

Figure 5

Formation of the INH-NADH adduct as catalyzed by WT KatG and resistance mutations in the presence of air-only (orange), 400 μM t-BuOOH (blue), 225 μM superoxide (red), 400 μM H₂O₂ administered over 1 h (black), 400 μM H₂O₂ administered over 3.3 h (gray), and 120 μM H₂O₂ administered over 1 h (cyan). The resistance mutations are color coded by their putative roles in KatG (refer to Fig. 3 and Table I). See “Experimental” section for reaction conditions. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

INH-resistance mutations, which give rise to significantly lower reactivity compared to WT KatG. A: Catalase activity attenuated ≥1000× (red). INH-NADH adduct formation attenuated ≥10× using: (B) 400 μM t-BuOOH (green), (C) 400 μM H₂O₂ over 1 h (G/GO_X, blue), (D) 225 μM (X/XO, pink). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]