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Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

ABSTRACT: The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.

SUBMITTER: Heym B

PROVIDER: S-EPMC204858 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

Heym B B Zhang Y Y Poulet S S Young D D Cole S T ST

Journal of bacteriology 19930701 13

The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to oth ...[more]

PMID: 8320241

Similar Datasets

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

Project description: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.

| S-EPMC6101688 | biostudies-literature

Antibiotic resistance in Mycobacterium tuberculosis: peroxidase intermediate bypass causes poor isoniazid activation by the S315G mutant of M. tuberculosis catalase-peroxidase (KatG).

Project description:KatG (catalase-peroxidase) in Mycobacterium tuberculosis is responsible for activation of isoniazid (INH), a pro-drug used to treat tuberculosis infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. The origin of INH resistance caused by the KatG[S315G] mutant enzyme is examined here. Overexpressed KatG[S315G] was characterized by optical, EPR, and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. Catalase activity and peroxidase activity with artificial substrates were moderately reduced (50 and 35%, respectively), whereas the rates of formation of oxyferryl heme:porphyrin pi-cation radical and the decay of heme intermediates were approximately 2-fold faster in KatG[S315G] compared with WT enzyme. The INH binding affinity for the resting enzyme was unchanged, whereas INH activation, measured by the rate of formation of an acyl-nicotinamide adenine dinucleotide adduct considered to be a bactericidal molecule, was reduced by 30% compared with WT KatG. INH resistance is suggested to arise from a redirection of catalytic intermediates into nonproductive reactions that interfere with oxidation of INH. In the resting mutant enzyme, a rapid evolution of 5-c heme to 6-c species occurred in contrast with the behavior of WT KatG and KatG[S315T] and consistent with greater flexibility at the heme edge in the absence of the hydroxyl of residue 315. Insights into the effects of mutations at residue 315 on enzyme structure, peroxidation kinetics, and specific interactions with INH are presented.

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Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: catalase, peroxidase, and INH-NADH adduct formation activities.

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| S-EPMC2866272 | biostudies-literature

Modeling the structural origins of drug resistance to isoniazid via key mutations in Mycobacterium tuberculosis catalase-peroxidase, KatG.

Project description:WHO reported 10.4 million new tuberculosis (TB) cases and 1.8 million deaths in 2015, making M. tuberculosis the most successful human pathogen with highest mortality among infectious diseases [1,2]. Drug-resistant TB is a major threat to global TB control [2,3]. Recently Torres et al. [4] identified 14 novel substitutions in M. tuberculosis-KatG (the enzyme associated with resistance to isoniazid-an important first-line anti-TB drug) and demonstrated that 12 of the 14 can cause INH-resistance in M. smegmatis. This study presents an in silico structure-based analysis of these 14 amino acid substitutions using homology models and x-ray crystal structures (when available) in M. tuberculosis. Our models demonstrate that several of these mutations cluster around three openings in the KatG tertiary structure which appear to initiate channels to the heme group at the catalytic center of the enzyme. We studied the effects of these mutations on the tertiary structure of KatG, focusing on conformational changes in the three channels in the protein structure. Our results suggest that the 14 novel mutations sufficiently restrict one or more of these access channels, thus potentially preventing INH from reaching the catalytic heme. These observations provide valuable insights into the structure-based origins of INH resistance and provide testable hypotheses for future experimental studies.

| S-EPMC7330162 | biostudies-literature

Access channel residues Ser315 and Asp137 in Mycobacterium tuberculosis catalase-peroxidase (KatG) control peroxidatic activation of the pro-drug isoniazid.

Project description:Peroxidatic activation of the anti-tuberculosis pro-drug isoniazid by Mycobacterium tuberculosis catalase-peroxidase (KatG) is regulated by gating residues of a heme access channel. The steric restriction at the bottleneck of this channel is alleviated by replacement of residue Asp137 with Ser, according to crystallographic and kinetic studies.

| S-EPMC3872143 | biostudies-literature

Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid.

Project description:Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.

| S-EPMC127408 | biostudies-literature

Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1.

Project description:A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.

| S-EPMC92299 | biostudies-literature

Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant <i>Mycobacterium tuberculosis</i> clinical isolate.

Project description:Mycobacterium tuberculosis catalase-peroxidase (Mt-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical M. tuberculosis isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG). The rate of IN-NAD formation mediated by WT-KatG was 23% greater than L333V-KatG when INH concentration is varied. In contrast to WT-KatG, the crystal structure of the L333V-KatG variant has a perhydroxy modification of the indole nitrogen of W107 from MYW adduct. L333V-KatG shows most of the active site residues in a similar position to WT-KatG; only R418 is in the R-conformation instead of the double R and Y conformation present in WT-KatG. L333V-KatG shows a small displacement respect to WT-KatG in the helix from R385 to L404 towards the mutation site, an increase in length of the coordination bond between H270 and heme Fe, and a longer H-bond between proximal D381 and W321, compared to WT-KatG; these small displacements could explain the altered redox potential of the heme, and result in a less active and stable enzyme.

| S-EPMC10839757 | biostudies-literature

Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis.

Project description:A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. The resultant enzymes were overexpressed, purified and characterized. Replacing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-275, which is probably involved in electron transfer from the haem, by proline resulted in a highly unstable enzyme with insignificant enzyme activities. The most commonly occurring substitution in drug-resistant clinical isolates is the replacement of Ser-315 by Thr; this lowered catalase and peroxidase activities by 50% and caused a significant decrease in the KatG-mediated inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier protein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of NitroBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro resulted in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.

| S-EPMC1220113 | biostudies-other

Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis.

Project description:Resolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding the binding mechanisms of small drug molecules. In Mycobacterium tuberculosis the multifunctional heme enzyme KatG is indispensable for activation of isoniazid (INH), a first-line pro-drug for treatment of tuberculosis. We present a cryo-EM methodology for structural and functional characterization of KatG and INH resistance variants. The cryo-EM structure of the 161 kDa KatG dimer in the presence of INH is reported to 2.7 Å resolution allowing the observation of potential INH binding sites. In addition, cryo-EM structures of two INH resistance variants, identified from clinical isolates, W107R and T275P, are reported. In combination with electronic absorbance spectroscopy our cryo-EM approach reveals how these resistance variants cause disorder in the heme environment preventing heme uptake and retention, providing insight into INH resistance.

| S-EPMC8355310 | biostudies-literature

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