Tuberculosis remains the largest cause of death in the world from a single infectious disease and is responsible for one in four avoidable adult deaths in developing countries. Infection with drug-sensitive strains of Mycobacterium tuberculosis can be effectively cured with a combination of isoniazid (INH), rifampicin, and pyrazinamide. However, the emergence of multiple drug resistant strains of M. tuberculosis has resulted in fatal outbreaks in the United States. INH was first reported to be active against M. tuberculosis in 1952, when it was shown to have a highly specific activity against M. tuberculosis and M bovis with less but considerable activity against other mycobacteria. Although INH is one of the most widely used anti-tuberculosis drugs for both therapy and prophylaxis, its precise target of action on Mycobacterium tuberculosis is unknown. We have discovered a novel gene, named inhA, in M. tuberculosis and all mycobacterial species examined that encodes a target for both isoniazid and ethionamide. The inhA gene encodes a protein of 32 kDa that shows significant sequence conservation with an E. coli enzyme, EnvM, known to play a role in fatty acid biosynthesis. Cell-free assays indicate the involvement of InhA protein in mycolic acid biosynthesis, previously suggested site of action of INH. In this proposal, we intend to employ a combination of genetic, biochemical, and x-ray crystallographic methods to characterize the InhA protein and elucidate mechanism of inhibition by ethionamide and isoniazid.
