Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis

• Published in: Biochemical journal Vol. 338; no. 3; pp. 753 - 760
• Main Authors: SAINT-JOANIS, Brigitte, SOUCHON, Hélène, WILMING, Martin, JOHNSSON, Kai, ALZARI, Pedro M., COLE, Stewart T.
• Format: Journal Article
• Language: English
• Published: Portland Press 08.03.1999
• Subjects: Amino Acid Sequence; Antitubercular Agents; Bacterial Proteins; Biochemistry, Molecular Biology; Bioinformatics; Biological Physics; Cellular Biology; Chemical Sciences; Computer Science; Cristallography; Disulfides; Enzyme Activation; Hydrolysis; Isoniazid; Kinetics; Life Sciences; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Nitroblue Tetrazolium; Peroxidases; Physics; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid; Structural Biology; prodrugs; mycolic acids; free radicals
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3380753

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.