Probing the function of Mycobacterium tuberculosis catalase-peroxidase by site-directed mutagenesis

2005 ◽  
pp. 3495 ◽  
Author(s):  
Nigel A. J. Eady ◽  
Jesmin ◽  
Spiros Servos ◽  
Anthony E. G. Cass ◽  
Judit M. Nagy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Catalase Peroxidase

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

1999 ◽  
Vol 338 (3) ◽  
pp. 753 ◽  
Author(s):  
Brigitte SAINT-JOANIS ◽  
Hélène SOUCHON ◽  
Martin WILMING ◽  
Kai JOHNSSON ◽  
Pedro M. ALZARI ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structure Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Catalase Peroxidase

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

1999 ◽  
Vol 338 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Brigitte SAINT-JOANIS ◽  
Hélène SOUCHON ◽  
Martin WILMING ◽  
Kai JOHNSSON ◽  
Pedro M. ALZARI ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Reduction Activity ◽  
Nitroblue Tetrazolium Reduction ◽  
Catalase Peroxidase ◽  
Tetrazolium Reduction

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.

Modulation of the Activities of Catalase−Peroxidase HPI ofEscherichia coliby Site-Directed Mutagenesis†

Biochemistry ◽  
2000 ◽  
Vol 39 (19) ◽  
pp. 5868-5875 ◽  
Author(s):  
Alex Hillar ◽  
Brian Peters ◽  
Ryan Pauls ◽  
Alexander Loboda ◽  
Haoming Zhang ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Catalase Peroxidase

scholarly journals Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C

2002 ◽  
Vol 367 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Radha CHAUHAN ◽  
Shekhar C. MANDE
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reaction Mechanism ◽  
Kinetic Parameters ◽  
Active Site ◽  
Double Mutant ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Resistant Strains

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.

scholarly journals Functional Analysis of DNA Gyrase Mutant Enzymes Carrying Mutations at Position 88 in the A Subunit Found in Clinical Strains of Mycobacterium tuberculosis Resistant to Fluoroquinolones

2006 ◽  
Vol 50 (12) ◽  
pp. 4170-4173 ◽  
Author(s):  
Stéphanie Matrat ◽  
Nicolas Veziris ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Chantal Truffot-Pernot ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Gyrase ◽  
Multidrug Resistant ◽  
Site Directed Mutagenesis ◽  
Fluoroquinolone Resistance ◽  
Directed Mutagenesis ◽  
The Novel ◽  
Wild Type ◽  
Clinical Strains ◽  
A Subunit

ABSTRACT We investigated the enzymatic efficiency and inhibition by quinolones of Mycobacterium tuberculosis DNA gyrases carrying the previously described GyrA G88C mutation and the novel GyrA G88A mutation harbored by two multidrug-resistant clinical strains and reproduced by site-directed mutagenesis. Fluoroquinolone MICs and 50% inhibitory concentrations for both mutants were 2- to 43-fold higher than for the wild type, demonstrating that these mutations confer fluoroquinolone resistance in M. tuberculosis.

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