Pyrazinamide (PZA) is an important component of the current short course tuberculosis (TB) chemotherapy. PZA plays a unique role in shortening the therapy to 6 months because it kills a population of semi-dormant tubercie bacilli that are not killed by other TB drugs. PZA is a prodrug that requires conversion to active form pyrazinoic acid by bacterial nicotinamidase/pyrazinamidase (PZase) to kill M. tuberculosis. We have identified the nicotinamidase/ PZase gene (pncA) and shown that mutations in pncA that affect PZA activation cause PZA resistance in tubercie bacillus. An intriguing observation from recent studies is that pncA mutations identified in PZA-resistant clinical isolates are highly diverse and scattered along the pncA gene. It has been difficult to correlate pncA mutations and the level of PZase activity with the level of resistance. We hypothesize that not all pncA mutations are equally important in affecting PZase activity and the level of PZA resistance. In this proposal, we propose to address this hypothesis using a combination of molecular genetic, biochemical and structural approaches.
The specific aims of the project are: (1) To characterize pncA mutations in PZA-resistant M. tuberculosis and assess their correlation with the level of PZase activity and PZA resistance. PZA-resistant clinical isolates of MTB will be analyzed in terms of pncA mutations and their correlation with the level of PZase activity and the level of resistance using more sensitive Cl 4-PZA PZase assay and PZA susceptibility testing method. (2) To identify the active site residues of PncA enzyme by site-directed mutagenesis. Catalytic site residues will be identified using PCR based site-directed mutagenesis with mutant oligonucleotide primers that alter desired amino acid residues. The mutant type PZase will be over expressed in E. coli and purified for PZase assay. (3) To carry out a full kinetic analysis of the pyrazinamidase (PZase) and nicotinamidase (NAMase) activities of wild type and mutant PncA enzymes. Enzyme kinetics for both purified wild type and mutant PncA enzymes will be determined and compared. The hypothesis that ferrous ion might enhance the enzyme activity by product removal will be tested. (4) To determine the crystal structure of wild type and mutant type PncA enzymes. Crystal structure of any PncA enzymes is unknown. The structure of wild type and mutant PncA enzymes will be determined to understand the paradox of how very diverse pncA mutations can all cause PZA resistance. These studies will provide important information about mechanisms of PZA action and resistance and may help design new antituberculosis drugs.
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