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.
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.