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

Mycobacterium tuberculosis isolates (n= 1,429) from 1,283 patients collected as part of an ongoing population-based tuberculosis epidemiology study in Houston, Texas, were analyzed by spoligotyping and IS6110 profiling. The isolates were also assigned to one of three major genetic groups on the basis of nucleotide polymorphisms located at codons 463 and 95 in the genes (katG and gyrA) encoding catalase-peroxidase and the A subunit of DNA gyrase, respectively. A total of 225 spoligotypes were identified in the 1,429 isolates. There were 54 spoligotypes identified among 713 isolates (n= 623 patients) assigned to 73 IS6110 clusters. In addition, among 716 isolates (n = 660 patients) with unique IS6110 profiles, 200 spoligotypes were identified. No changes were observed either in the IS6110 profile or in the spoligotype for the 281 isolates collected sequentially from 133 patients. Five instances in which isolates with slightly different spoligotypes had the same IS6110 profile were identified, suggesting that in rare cases isolates with different spoligotypes can be clonally related. Spoligotypes correlated extremely well with major genetic group designations. Only three very similar spoligotypes were shared by isolates from genetic groups 2 and 3, and none was shared by group 1 and group 2 organisms or by group 1 and group 3 organisms. All organisms belonging to genetic groups 2 and 3 failed to hybridize with spacer probes 33 to 36. Taken together, the results support the existence of three distinct genetic groups of M. tuberculosis organisms and provide new information about the relationship between IS6110 profiles, spoligotypes, and major genetic groups of M. tuberculosis.

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ethA, inhA, and katG Loci of Ethionamide-Resistant Clinical Mycobacterium tuberculosis Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.12.3799-3805.2003 ◽

2003 ◽

Vol 47 (12) ◽

pp. 3799-3805 ◽

Cited By ~ 159

Author(s):

Glenn P. Morlock ◽

Beverly Metchock ◽

David Sikes ◽

Jack T. Crawford ◽

Robert C. Cooksey

Keyword(s):

Mycobacterium Tuberculosis ◽

Structural Gene ◽

Single Point ◽

Resistance Mechanism ◽

Structural Analog ◽

Mycolic Acid ◽

Single Point Mutation ◽

Structural Genes ◽

Resistance Mutations ◽

Catalase Peroxidase

ABSTRACT Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 μg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were ≥100 μg/ml, and these isolates were also resistant to ≥8 μg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were ≥32 μg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to ≥50 μg of ETH per ml had such mutations.

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Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

Journal of Bacteriology ◽

10.1128/jb.186.16.5427-5431.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5427-5431 ◽

Cited By ~ 30

Author(s):

Graham S. Timmins ◽

Sharon Master ◽

Frank Rusnak ◽

Vojo Deretic

Keyword(s):

Nitric Oxide ◽

Mycobacterium Tuberculosis ◽

Reactive Species ◽

Front Line ◽

Respiratory Enzymes ◽

Catalase Peroxidase ◽

Oxidative Activation

ABSTRACT Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO˙) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro. We also provide in vivo evidence that INH-derived NO˙ can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.

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Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T

Journal of Inorganic Biochemistry ◽

10.1016/j.jinorgbio.2005.03.016 ◽

2005 ◽

Vol 99 (6) ◽

pp. 1401-1406 ◽

Cited By ~ 7

Author(s):

Sofia M. Kapetanaki ◽

Salem Chouchane ◽

Shengwei Yu ◽

Richard S. Magliozzo ◽

Johannes P.M. Schelvis

Keyword(s):

Raman Spectroscopy ◽

Mycobacterium Tuberculosis ◽

Resonance Raman Spectroscopy ◽

Resonance Raman ◽

Resistant Mutant ◽

Catalase Peroxidase ◽

Compound Ii

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Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2137-2144.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2137-2144 ◽

Cited By ~ 50

Author(s):

Michel Nguyen ◽

Annaïk Quémard ◽

Sylvain Broussy ◽

Jean Bernadou ◽

Bernard Meunier

Keyword(s):

Mycobacterium Tuberculosis ◽

Biosynthetic Pathway ◽

Acyl Carrier Protein ◽

Isonicotinic Acid ◽

Structural Investigations ◽

Mycolic Acids ◽

Nicotinamide Mononucleotide ◽

Catalase Peroxidase ◽

Nicotinamide Coenzymes

ABSTRACT The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.

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Antibiotic Resistance in Mycobacterium tuberculosis

Journal of Biological Chemistry ◽

10.1074/jbc.m109.005546 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16146-16155 ◽

Cited By ~ 17

Author(s):

Javier Suarez ◽

Kalina Ranguelova ◽

Johannes P. M. Schelvis ◽

Richard S. Magliozzo

Keyword(s):

Mycobacterium Tuberculosis ◽

Resonance Raman Spectroscopy ◽

Rapid Evolution ◽

Cation Radical ◽

Artificial Substrates ◽

Mutant Enzyme ◽

Activation Mechanism ◽

Catalytic Intermediates ◽

Catalase Peroxidase

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 π-cation radical and the decay of heme intermediates were ∼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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