Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis.
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.
Saint-Joanis
B
B
Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Souchon
H
H
Wilming
M
M
Johnsson
K
K
Alzari
P M
PM
Cole
S T
ST
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D006868:Hydrolysis; D007538:Isoniazid / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D009580:Nitroblue Tetrazolium; D010544:Peroxidases; C076136:catalase HPI; D007538:Isoniazid