Comparative characterization and structure-function analyses of the catalase-peroxidases of Escherichia coli and Mycobacterium tuberculosis
Hillar, Alexander Peter
Both 'Escherichia coli' and 'Mycobacterium tuberculosis ' produce catalase-peroxidases encoded by the katG structural gene as part of their cellular defence systems against oxidative stress. The katG gene products, KatG or BPI (Hydroperoxidase I; an alternate name for the ' E. coli' enzyme) have high catalase activities, as well as low level peroxidase activities utilizing organic electron donors. The catalase-peroxidase from 'M. tuberculosis', KatG or MtHPI, has been implicated in the mode of action of the antitubercular drug isoniazid (isonicotinic: acid hydrazide, or INH), whereby MtHPI oxidizes INH to an electrophilic form capable of irreversibly binding enzymes involved in mycolic acid synthesis in the cells. Characterization of certain biochemical and structural properties of recombinant MtHPI and the homologous recombinant 'E. coli' catalase-peroxidase (EcHPI) was carried out, in order to determine whether differences in the enzymes could be partially responsible for lack of INH susceptibility in 'E.coli' compared to 'M. tuberculosis '. MtHPI and EcHPI were similar with regard to subunit sizes and their intrinsic inhomogeneity estimated by SDS-PAGE analysis and mass spectrometry, affinity for cyanide as a heme ligand, and susceptibility to cyanide and azide as enzyme inhibitors. In contrast, MtHPI had higher peroxidase and lower catalase activity than EcHPI, and was more proficient at both INH oxidation (k cat of 8.7 * 104 s-1 compared to 2.4 * 104 s-1 for EcHPI) and INH binding (Kd of 17 [mu]M INH compared to 130 [mu]M for EcHPI), determined using spectrophotometric techniques. The intracellular locale of (EcHPI) in ' E. coli' was also determined as a second major part of this study. Assays of catalase and corroborating enzymatic activities in fractions prepared via spheroplasting procedures, as well as in situ immunogold staining followed by microscopic analysis, confirmed that (EcHPI) occurs predominantly in the cytoplasm of 'E. coli' cells, contrary to a previous report of EcHPI being a periplasmic enzyme. A structure-function study of EcHPI via site-directed mutagenesis was carried out as a third principal part of this study, focusing mainly on certain amino acid residues in the putative active site of the enzyme. Changing the His106, Arg102, Trp105, and His267 residues in EcHPI resulted in significant changes in enzymatic activities as well as changes in absorption spectra, heme content, susceptibility to inhibitors, and cyanide binding in the mutant variant enzymes. Changes to His 106 and His267 resulted in drastically decreased catalatic and peroxidatic activities, providing evidence that these residues are critical to enzyme catalysis. Changes to Arg102 resulted in decreased catalatic and peroxidatic activities, however the changes in activity were not as drastic as those for the His106 and His267 variants, suggesting that Arg102 is important but not critical to enzyme catalysis. Changes to Trp105 resulted in catalatic activity decreased by three orders of magnitude, but either an increase (Trp105Phe) or no change (Trp105Leu) in the peroxidatic activity of these mutant variants. Investigation of the Trp105, mutant variants also showed that both are capable of forming a spectral species similar to the compound In intermediate of plant peroxidases, and that this species is rapidly inactivated, in the presence of hydrogen peroxide. A novel catalatic reaction mechanism is proposed for catalase-peroxidases based on active site models of peroxidases and the information derived for the modulation of activities that occurs for the Trp105 mutant variants.