The goals of this project are to define catalytic functions of the enzyme catalase-peroxidase (KatG) from M. tuberculosis relevant to the mechanism of action of the antituberculosis antibiotic isoniazid (INH). INH has been used for over fifty years as one of the most potent therapeutic agents against tuberculosis infection but continuing development of resistance to it and other drugs warrants new research into the biology of the TB pathogen, M. tuberculosis, the mode of action of INH and the origins of drug resistance. The activation of INH depends on the catalytic functions of the enzyme KatG but details of this mechanism are not well understood. Most INH resistant bacteria isolated from patients throughout the world who do not respond to treatment by this drug carry a mutation in the gene coding for the KatG enzyme. The single amino acid mutations presumably alter enzyme structure and the function of catalytic intermediates required for activation of INH. In the proposed research the tools of enzymology, spectroscopy and structural biology will be applied to probe the structure of KatG enzymes and how amino acid replacements interfere with antibiotic activation. The research will use rapid kinetics measurements based on optical stopped-flow spectrophotometry and rapid freeze-quench electron paramagnetic resonance spectroscopy, and will also apply isothermal titration calorimetry, X-ray crystallography, resonance Raman spectroscopy and chemical calculations focusing on enzyme structure, function and key interactions between the enzyme and the antibiotic. Both the catalase and the peroxidase reaction pathways operating in KatG will be investigated. The specific role of catalytically competent intermediates formed when the enzyme reacts with peroxide will be investigated to probe their function and to compare the properties of the wild-type enzyme with those of mutant enzymes. The research will provide insights into the fundamental origins of a worldwide health problem that affects millions of people each year.

Public Health Relevance

The project is devoted to explaining the origins of antibiotic (isoniazid) resistant tuberculosis infection. The focus is on the structure and function of catalase-peroxidase, a heme enzyme in M. tuberculosis responsible for activation of isoniazid, and the faulty activation of the antibiotic due to mutations in this enzyme found in clinical strains resistant to treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI060014-06A1
Application #
8145057
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Lacourciere, Karen A
Project Start
2004-01-01
Project End
2013-08-31
Budget Start
2010-09-23
Budget End
2013-08-31
Support Year
6
Fiscal Year
2010
Total Cost
$509,394
Indirect Cost
Name
Brooklyn College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
620127691
City
New York
State
NY
Country
United States
Zip Code
11210
Teixeira, Vitor H; Ventura, Cristina; Leitão, Ruben et al. (2015) Molecular details of INH-C10 binding to wt KatG and Its S315T mutant. Mol Pharm 12:898-909
Zhao, Xiangbo; Hersleth, Hans-Petter; Zhu, Janan et al. (2013) Access channel residues Ser315 and Asp137 in Mycobacterium tuberculosis catalase-peroxidase (KatG) control peroxidatic activation of the pro-drug isoniazid. Chem Commun (Camb) 49:11650-2
Zhao, Xiangbo; Khajo, Abdelahad; Jarrett, Sanchez et al. (2012) Specific function of the Met-Tyr-Trp adduct radical and residues Arg-418 and Asp-137 in the atypical catalase reaction of catalase-peroxidase KatG. J Biol Chem 287:37057-65
Zhao, Xiangbo; Yu, Shengwei; Ranguelova, Kalina et al. (2009) Role of the oxyferrous heme intermediate and distal side adduct radical in the catalase activity of Mycobacterium tuberculosis KatG revealed by the W107F mutant. J Biol Chem 284:7030-7
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Suarez, Javier; Ranguelova, Kalina; Jarzecki, Andrzej A et al. (2009) An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG). J Biol Chem 284:7017-29
Suarez, Javier; Ranguelova, Kalina; Schelvis, Johannes P M et al. (2009) Antibiotic resistance in Mycobacterium tuberculosis: peroxidase intermediate bypass causes poor isoniazid activation by the S315G mutant of M. tuberculosis catalase-peroxidase (KatG). J Biol Chem 284:16146-55