Three-Dimensional Model and Molecular Mechanism ofMycobacterium tuberculosisCatalase-Peroxidase (KatG) and Isoniazid-Resistant KatG Mutants

Abstract

Mycobacterium tuberculosis KatG enzyme functions both as catalase for removing hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 binding site. This H 2 O 2 binding site appears to interact with the catalytic site by a possible electron-transfer chain, a Met255-Tyr229-Trp107 triad conserved in many catalaseperoxidases. 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.

A molecular switch and electronic circuit modulate catalase activity in catalase‐peroxidases

“…Similar to changes to the Trp, changes to the Tyr or Met, or even Arg 426 associated with Tyr 238, inhibited the catalase reaction, with little or no effect on the peroxidase reaction (Jakopitsch et al, , 2004Yu et al, 2003;Singh et al, 2004). A mechanism attempting to explain why these residues are essential for the catalase reaction has been postulated (Mo et al, 2004), but is incompatible with recent data. a BpKatG_7.5, crystal at pH 7.5; BpKatG_8.0, crystal at pH 8.0; OF_5.6, oxyferryl species at pH 5.6; OF_7.5, oxyferryl species (generated at pH 5.6 and shifted to pH 7.5) from the composite data set described in Methods.…”

“…Similar to changes to the Trp, changes to the Tyr or Met, or even Arg 426 associated with Tyr 238, inhibited the catalase reaction, with little or no effect on the peroxidase reaction (Jakopitsch et al , 2003, 2004; Yu et al , 2003; Singh et al , 2004). A mechanism attempting to explain why these residues are essential for the catalase reaction has been postulated (Mo et al , 2004), but is incompatible with recent data.…”

Novel mutations in katG gene of a clinical isolate of isoniazid-resistant Mycobacterium tuberculosis

“…Even though katG (S315T) has catalase and peroxidase activities, it is less efficient than KatGWT in the isoniazid metabolism [ 14 ]. Modification of the INH binding site due to the S315T mutation is a significant factor in the decline of the mutant activity to activate isoniazid [ 15 ] [ 16 ].…”

Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 catalase-peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy

“…Although the KatG active sites globally resembles that of mono-functional peroxidases, in the detail there are significant differences on the heme environment. Recent investigations based on structural aspects [2,3,12,13] as well as kinetic characterization of selected variants of the heme environment [14][15][16][17]19] have given the basis to better understand the contribution of such differences in the remarkable reactivity of KatGs, as compared to mono-functional peroxidases. For example, it has been shown that the very unusual adduct resulting from the crosslink of three residues of the heme distal side, namely Trp122, Tyr249 and Met275 (Synechocystis numbering) is crucial for the catalatic activity of KatGs.…”