Three-Dimensional Model and Molecular Mechanism of Mycobacterium tuberculosis Catalase-Peroxidase (KatG) and Isoniazid-Resistant KatG Mutants

Abstract

Mycobacterium tuberculosis KatG enzyme functions both as catalase for removing hydrogen peroxide (H₂O₂) and as peroxidase for oxidating isoniazid (INH) to active form of anti-tuberculosis drug. Although mutations in M. tuberculosis KatG confer INH resistance in tuberculous patients, structural bases for INH-resistant mutations in the KatG gene remains poorly understood. Here, three M. tuberculosis KatG mutants bearing Arg418→ Gln, Ser315 → Thr, or Trp321 → Gly replacement were assessed for changes in catalase-peroxidase activities and possible structure bases relevant to such changes. These three M. tuberculosis KatG mutants exhibited a marked impairment or loss of catalase-peroxidase activities. The possible structural bases for the mutant-induced loss of enzyme activities were then analyzed using a three-dimensional model of M. tuberculosis KatG protein constructed on the basis of the crystal structure of the catalase-peroxidase from Burkholderia pseudomallei. The model suggests that three M. tuberculosis KatG mutants bearing Arg418 → Gln, Ser315 →Thr, or Trp321→ Gly replacement affect enzyme activities by different mechanisms, although each of them impacts consequently on a heme-associated structure, the putative oxidative site. Moreover, in addition to the widely accepted substrate-binding site, M. tuberculosis KatG may bear another H₂O₂ binding site. This H₂O₂ binding site appears to interact with the catalytic site by a possible electron-transfer chain, a Met255-Tyr229-Trp107 triad conserved in many catalase-peroxidases. The Ser315 → Thr mutant may have direct effect on the catalytic site by interfering with electron transfer in addition to the previously proposed mechanism of steric constraint.