Isoniazid activation defects in recombinant Mycobacterium tuberculosis catalase-peroxidase (KatG) mutants evident in InhA inhibitor production

Abstract

Mycobacterium tuberculosis KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical Mycobacterium tuberculosis isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatG(A110V), KatG(A139P), KatG(S315N), KatG(L619P), KatG(L634F), and KatG(D735A)) were purified and characterized with respect to their catalase-peroxidase activities (in terms of k(cat)/K(m)), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatG(L634F), KatG(A139P), and KatG(D735A) variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatG(S315N) and KatG(L619P) variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn(2+) to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of wild-type and mutant KatGs. Lanes: M, molecular size markers; 1, wild type; 2, D735A; 3, A110V; 4, S315N; 5, A139P; 6, L619P, 7; L634F.

FIG. 2.

KatG-mediated oxidation of isoniazid, followed by NBT reduction. All experiments were carried out at room temperature. To 1 ml of 50 mM phosphate buffer, KatG proteins, NBT, glucose oxidase, and glucose were added at 1 μM, 0.2 mM, 5 mg, and 4 mM, respectively. (A) Time course of NBT reduction with and without the addition of 9 mM isoniazid (INH) with wild-type KatG. (B) Time courses of net isoniazid-dependent NBT reduction by wild-type KatG (WT) and three variants. For each KatG sample tested, NBT reduction in the absence of isoniazid was subtracted from that in the presence of isoniazid to obtain the net isoniazid-dependent NBT reduction over time.

FIG. 3.

Isoniazid activation by wild-type and mutant KatG. The reactions were carried out for 3 h at room temperature with a sample of 1 ml of 50 mM phosphate, pH 7.0, containing 1 mM isoniazid, 1 mM NADH, and 0.5 μM wild-type KatG or a A110V, A139P, S315N, L619P, L634F, or D735A KatG mutant. KatG was then removed with Micron YM-10 filter units. One milliliter of sample containing 0.8 ml of filtrate and 0.2 ml of 78.5 μM InhA was incubated at room temperature for 20 min. The mixture was subjected to Sephadex G-25 column filtration twice, and the InhA-inhibitor complex was isolated. The spectra were normalized to a content of 10 μM InhA. (A) Absorption spectrum of the InhA-inhibitor complex with wild-type KatG for isoniazid activation. (B) Absorption spectrum of the same InhA-inhibitor complex derived from wild-type KatG (WT) activation of isoniazid is reproduced for the range of 300 to 400 nm. This spectrum is compared with those obtained under identical conditions with either the L634F or S315N KatG mutant in place of wild-type KatG. The final spectrum obtained for a negative control, in which wild-type KatG was used but no isoniazid was added, under otherwise identical conditions was identical to that shown for the S315N KatG mutant. For simplicity, spectra obtained with the other KatG mutants are not included. The corresponding yields of the inhibitor derived from isoniazid activation by all KatG species tested were calculated from the spectral data so obtained and are presented in Table 4.

FIG. 4.

Comparison of enzyme activities of KatG mutants with wild-type KatG. Results of catalase (○), peroxidase (□), isoniazid oxidation/NBT reduction (▵), and isoniazid activation in the absence of Mn²⁺ (•) activity determinations described in the text were used to calculate the relative activities, defined as the ratios of KatG mutant activity to wild-type KatG activity.