Radical formation and functions in Mycobacterium tuberculosis catalase-peroxidase (KatG) and in Cryptococcus neoformans melanin
Item
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Title
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Radical formation and functions in Mycobacterium tuberculosis catalase-peroxidase (KatG) and in Cryptococcus neoformans melanin
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Identifier
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d_2009_2013:fbcebc0164f5:11505
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identifier
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11994
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Creator
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Khajo, Abdelahad,
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Contributor
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Richard S. Magliozzo
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Pharmacy sciences | Biochemistry | Biophysics
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Abstract
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The dual-function heme enzyme catalase-peroxidase, KatG, is found in many microorganisms but in the human pathogen M. tuberculosis it is the only catalase (2H2O2 → O2 + 2H2O) and plays an important role in protecting this organism against oxidative stress. The structural and functional origins of its high catalase activity are therefore of special interest. KatG also exhibits a broad-spectrum peroxidase activity (ROOH + 2AH → 2ROH + 2A· ) and in M. tuberculosis it is responsible for peroxidative activation of the pro-drug isoniazid used to treat tuberculosis infection.;Previous studies have shown that the catalase activity of KatG depends upon a unique adduct structure consisting of three conserved amino acids (Met255, Tyr229 and Trp107, M-Y-W) on the distal side of the heme pocket and that this adduct enables formation of a unique radical intermediate required for efficient turnover of H2O2. Here, EPR measurements along with site-directed mutagenesis and isotope labeling experiments allowed assignment of the 'narrow doublet' radical signal found during catalase turnover to Tyr229 of the M-Y-W adduct, explained the operation of this radical in KatG using optical and stopped-flow spectrometry, and elucidated a function for this unique cofactor and a role for neighboring residues in the enzyme's catalase activity. Based on these findings, a new radical-dependent catalase reaction mechanism in KatG could be proposed.;Previous studies also showed formation of other amino acid-based radicals in KatG upon reaction with alkyl peroxides. Gaining insights into the poorly-understood peroxidase reaction mechanism in KatG through identifying the location and the possible function of the 'wide doublet' radical signal was among the goals of this thesis. Mutagenesis and rapid freeze-quench EPR experiments revealed the involvement of several residues in an electron transfer pathway including Tyr229, Tyr597, Tyr678, Trp90, Trp135, and Trp149. Other important aspects regarding the peroxidase function of KatG such as the electronic structure of its initial hypervalent heme species and the role of proximal Trp321 were studied. While details of the peroxidase reaction in KatG are far from being resolved, a mechanism was presented based on the available data.;The second part of this work was devoted to studying another biologically-relevant free radical, which occurs in eumelanin from the fungus C. neoformans. With the application of EPR spectroscopy, UV-Vis spectrometry, chemical analyses, and other physico-chemical techniques to investigate how high-dose gamma irradiation under aqueous aerobic conditions interacts with the semiquinone radical and affects the physical and chemical properties of natural and synthetic melanins, the studies aimed to gain insights into a radioprotective role of melanin in fungal cells.;Findings here revealed a shielding function for melanin in live fungal cells from high-dose ionizing radiation by attenuating the destructive effects of radiolysis-induced hydroxyl radicals and serving as a sacrificial barrier in the cell wall. Chemical analysis of irradiated samples showed that hydroxyl radical attack on susceptible sites in melanin subunits lead to C-C bond cleavage and the release of low molecular weight aldehydes.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Chemistry
