Probing the function of Mycobacterium tuberculosis catalase-peroxidase by site-directed mutagenesis.
Catalase-peroxidase is a multi-functional heme-dependent enzyme which is well known for its ability to carry out both catalatic and peroxidatic reactions. Catalase-peroxidase from Mycobacterium tuberculosis(mtCP) is of particular interest because this enzyme activates the pro-antitubercular drug isoniazid. It is estimated that 2 billion people are infected with M. tuberculosis, the principal causative agent of tuberculosis, and that 2 million people die from the disease each year. The rise of drug-resistant strains continues to be of critical concern and it is well documented that mutations which reduce activity or inactivate mtCP lead to increased levels of isoniazid resistance in M. tuberculosis. The recent determination of the crystal structure for M. tuberculosis mtCP has aided the understanding of how the enzyme functions and provides a three-dimensional framework for testing hypotheses about the roles of various residues in the active site. Here we report site-directed mutagenesis studies of three conserved residues located near the heme of mtCP, His-108, Trp-107 and Trp-321 including the construction of the double mutant W107F-W321F. Resulting mutants have been purified and their catalatic and peroxidatic activities have been determined. Data are compared in the context of related studies aimed at dissecting the roles of these residues in the different activities of the enzyme. Analyses of single and double mutants studied here emphasise that the hydrogen bonding network surrounding the heme in the active site appears more important for maintenance of catalatic rather than peroxidatic activity in CP enzymes.
Eady
Nigel A J
NA
Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, UK.
Jesmin
Nigel A J
NA
Servos
Spiros
S
Cass
Anthony E G
AE
Nagy
Judit M
JM
Brown
Katherine A
KA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria