OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CATALASE-PEROXIDASE KATG FROM MYCOBACTERIUM-TUBERCULOSIS

Wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resist ant strains have been overexpressed in Escherichia coli, allowing puri fication of sufficient quantities of enzyme for physical and kinetic c haracterization. Optical absorption and EPR spectroscopies indicate th at KatG is similar to a growing class of bacterial catalase-peroxidase s. Optical and EPR spectra of KatG in the presence of either a strong field or weak field ligand suggest that, like horseradish peroxidase a nd metmyoglobin, KatG is likely to have a histidine as a proximal liga nd. The wild-type enzyme functions as a highly active catalase as well as a broad specificity peroxidase. Wild-type KatG and the R463L mutan t of KatG exhibit identical spectroscopic and kinetic properties. Furt hermore, both enzymes are equally capable of metabolizing the importan t antituberculosis drug isoniazid.