A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. The resultant enzymes were overexpressed, purified and characterized. Replacing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-275, which is probably involved in electron transfer from the haem, by proline resulted in a highly unstable enzyme with insignificant enzyme activities. The most commonly occurring substitution in drug-resistant clinical isolates is the replacement of Ser-315 by Thr; this lowered catalase and peroxidase activities by 50% and caused a significant decrease in the KatG-mediated inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier protein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of NitroBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro resulted in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.
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March 1999
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Research Article|
March 08 1999
Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis Available to Purchase
Brigitte SAINT-JOANIS;
Brigitte SAINT-JOANIS
*Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
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Hélène SOUCHON;
Hélène SOUCHON
†Unité de Biochimie Structurale, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
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Martin WILMING;
Martin WILMING
‡Lehrstuhl für Organische Chemie I, Ruhr-Universität Bochum, 44780 Bochum, Germany
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Kai JOHNSSON;
Kai JOHNSSON
‡Lehrstuhl für Organische Chemie I, Ruhr-Universität Bochum, 44780 Bochum, Germany
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Pedro M. ALZARI;
Pedro M. ALZARI
†Unité de Biochimie Structurale, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
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Stewart T. COLE
Stewart T. COLE
1
*Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
September 29 1998
Revision Received:
November 06 1998
Accepted:
December 16 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 338 (3): 753–760.
Article history
Received:
September 29 1998
Revision Received:
November 06 1998
Accepted:
December 16 1998
Citation
Brigitte SAINT-JOANIS, Hélène SOUCHON, Martin WILMING, Kai JOHNSSON, Pedro M. ALZARI, Stewart T. COLE; Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis. Biochem J 15 March 1999; 338 (3): 753–760. doi: https://doi.org/10.1042/bj3380753
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