Molecular Analysis of katG Encoding Catalase-Peroxidase from Clinical Isolate of Isoniazid-Resistant Mycobacterium tuberculosis

Abstract

Isoniazid (INH) is a drug for the treatment of tuberculosis in patients infected with Mycobacterium tuberculosis. The katG enzyme, or catalase-peroxidase, activates the pro-drug INH that is coded by the katG gene in M. tuberculosis. Mutations of the katG gene in M. tuberculosis are a major INH resistance mechanism. The M. tuberculosis clinical isolate R2 showed INH resistance at a high level of 10 µg/mL. However, the molecular basis for the resistance is unclear. The identification of a mutation in the katG gene of the clinical isolate R2 showed four mutations, i.e., C1061T, G1261 A, G1388T, G2161A, which correspond to the amino acid substitutions T354I, G421S, R463L, and V721M, respectively. The mutant katG gene, along with the wild-type were cloned, expressed and purified. The mutant enzyme showed 86.5% of catalase and 45% of peroxidase activities in comparison to the wild type. The substitutions of T₃₅₄I and G₄₂₁S in mutant katG R2 created significant instability in the adduct triad complex (Trp107-Tyr229-Met255), a part of the active site of the catalase-peroxidase enzyme in the model structure analysis. The events could be based on the high resistance of the clinical isolate R2 toward INH as the molecular basis.

Keywords: Mycobacterium tuberculosis; catalase-peroxidase; isoniazid; katG.

Fig. 1

PCR product in agarose gel electrophoresis. M, Marker DNA λ/HindIII (M); lane 1 and 2, represented the DNA fragment (2,2 kb) that resulted by PCR using the genome of M. tuberculosis H37RV and INH-resistant M. tuberculosis R2 as templates respectively. The DNA fragment of 2.2 kb corresponds to the katG gene.

Fig. 2

The pT7-Blue-katG recombinant in agarose gel electrophoresis. M, marker DNA λ/HindIII; lane 1, pT7Blue-katG M. tuberculosis H37RV/ NdeI+XbaI; lane 2-3, pT7Blue-katG R2/ NdeI+XbaI. The pT7Blue had a size of 2.9 kb, then katG had a size of 2,2 kb.

Fig. 3

Electrophoretogram of sequence katG R2 toward katG H37RV and Genbank. KatG R2 had four mutations, C₁₀₆₁T, G₁₂₆₁ A, G₁₃₈₈T, G₂₁₆₁A compared to katG H37RV.

Fig. 4

The pCold II-katG recombinant in agarose gel electrophoresis. M, marker DNA λ/Hind III; 1, restriction of pcold II-katG recombinant with Nde I and XbaI resulted in fragments of 4.2 kb and 2.2 kb belonging to pCold II-DNA and katG respectively.

Fig. 5

KatG protein in SDS-PAGE. M (marker), 1, crude protein of katG; 2, flow throw; 3-4 washing; 5, protein katG WT; 6-7, protein katG R2 after eluted with 150 mM imidazole

Fig. 6

The catalase activity of katG. The mutant katG R2 had a catalase activity of 86.5% of the wild-type activity.

Fig. 7

The peroxidase activity of katG. The mutant katG R2 had a peroxidase activity of 45% of its wild-type activity.

Fig. 8

Illustration of effect Gly421Ser, Arg463Leu substitution in katG R2 structure model. (A) The wild-type katG structure with Thr354 polar residue in the catalytic site region (green represents amino acid residues). (B) The mutant katG structure L10 that carried Ile354 residue. The Thr354Ile substitution eliminated the interaction between Thr354 and Thr376. (C) The wild-type katG structure with a stable interaction in the adduct triad complex (a magnetized rod-shaped residue of Ser421 residue causing a new interaction linking the Ser421 residue to the Arg489 residue). This new interaction is not found in the katG WT structure.