Tuberculosis (TB) is a global health emergency that remains the leading cause of human mortality due to a bacterial pathogen, the Mycobacterium tuberculosis. It is estimated that 8.5 million people are infected and 2 million die annually, and more than 95% of these deaths occur in developing countries. The long treatment-time, based on a four-drug regime of isoniazid, rifampicin, pyrazinamide, and ethambutol (or streptomycin), the co-infection with HIV, and the generation of multi-drug resistant strains (MDR-TB), makes the development and use of new drugs against M. tuberculosis extremely necessary. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of the aromatic amino acids tyrosine, tryptophane and phenylalanine. This pathway is present in bacteria, algae, fungi, plants and parasites belonging to phylum Apicomplexa, but is absent in mammals. Prephenate dehydrogenase (TyrA) is the penultimate enzyme involved in the biosynthesis of tirosyne, and might represent a potential target for the development of new anti-TB drugs. This work reports the cloning of the tyrA gene from M. tuberculosis H37Rv and the expression of the encoded recombinant enzyme. The gene was amplified by PCR and subcloned into pET23a(+) expression vector, and the clone was sequenced to confirm that no mutations were inserted during the amplification and cloning steps. The recombinant enzyme was expressed in E. coli BL21(DE3) host cells, although in the insoluble fraction. Soluble enzyme expression, purification and biochemical characterization of M. tuberculosis TyrA are currently in progress.

Supported by BPA/PUC