The major role of the propose research is to explain the catalytic function of Mycobacterium Tuberculosis catalase-peroxidase, a heme-enzyme, in the activation of the antimycobacterial antibiotic, isoniazid (isonicotinic acid hydrazide).
The specific aims i nclude identification of the proximal ligand to heme iron in catalase-peroxidase, characterization of the spin state and coordination number of the heme iron in the resting enzyme, identification of hypervalent enzyme intermediates, and kinetic analysis of the reaction of these intermediates with isoniazid. Of special interest is the potential catalytic competence of oxy-ferrous catalase- peroxidase, and the potential for peroxynitrite to activate the enzyme. The role of selected amino acid residues in the catalytic mechanism an in isoniazid binding will be evaluated through examination of the properties of two mutant catalase-peroxidase enzymes identified from clinically isolated, isoniazid resistant M. tuberculosis strains. Inhibition of another M. tuberculosis enzyme, a fatty acyl enoyl reductase (the inhA protein) thought to be a target of drug action, by isoniazid activated catalase-peroxidase, will also be investigated.
Other aims address the Mn(II)-peroxidase activity of catalase-peroxidase considered important because Mn(III) is an efficient single electron oxidant of isoniazid. The techniques of optical stopped-flow spectroscopy, resonance Raman, and electron paramagnetic resonance spectroscopies will be applied in the experimental protocols. The results of the proposed studies will advance a detailed understanding of the action of a first line antibiotic in current use to treat tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI043582-02
Application #
6012843
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1998-06-15
Project End
2003-05-31
Budget Start
1999-01-01
Budget End
1999-05-31
Support Year
2
Fiscal Year
1998
Total Cost
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
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
Ranguelova, Kalina; Suarez, Javier; Magliozzo, Richard S et al. (2008) Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase. Biochemistry 47:11377-85
Ranguelova, Kalina; Girotto, Stefania; Gerfen, Gary J et al. (2007) Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings. J Biol Chem 282:6255-64
Zhao, Xiangbo; Yu, Hong; Yu, Shengwei et al. (2006) Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant. Biochemistry 45:4131-40
Kapetanaki, Sofia M; Chouchane, Salem; Yu, Shengwei et al. (2005) Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function. Biochemistry 44:243-52
Kapetanaki, Sofia M; Chouchane, Salem; Yu, Shengwei et al. (2005) Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T. J Inorg Biochem 99:1401-6
Zhao, Xiangbo; Girotto, Stefania; Yu, Shengwei et al. (2004) Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG). J Biol Chem 279:7606-12
Yu, Shengwei; Girotto, Stefania; Lee, Chiuhong et al. (2003) Reduced affinity for Isoniazid in the S315T mutant of Mycobacterium tuberculosis KatG is a key factor in antibiotic resistance. J Biol Chem 278:14769-75
Yu, Shengwei; Girotto, Stefania; Zhao, Xiangbo et al. (2003) Rapid formation of compound II and a tyrosyl radical in the Y229F mutant of Mycobacterium tuberculosis catalase-peroxidase disrupts catalase but not peroxidase function. J Biol Chem 278:44121-7
Kapetanaki, Sofia; Chouchane, Salem; Girotto, Stefania et al. (2003) Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy. Biochemistry 42:3835-45

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