Biochemical analysis of the Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX

Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new drugs. Thymidylate synthase (TS) enzymes from other species are considered good targets for drug development and the M. tuberculosis genome contains two TS enzymes, the conventional ThyA and the flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug resistance studies or genome-wide mutagenesis screens.;After overexpression and purification, the thymidylate synthesizing abilities of M. tuberculosis ThyA and ThyX were verified and a turnover-dependent tritium- release assay was used to characterize their reaction kinetics. While both enzymes bound their proposed substrates with similar affinities to analogous enzymes, they displayed differential binding to folate and nucleotide-based inhibitors. Several potent inhibitors of human ThyA display very low inhibitory activity against the M. tuberculosis enzymes. The unique ligand-binding properties of these enzymes present an opportunity for the design of species specific and enzyme selective inhibitors. Additional studies using M. tuberculosis ThyX showed that its reaction mechanism is highly different from the classical ThyA further highlighting its unique features.;An active-site titration method revealed that both M. tuberculosis enzymes had very low kcat values. One possible explanation for the low catalytic activity of ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, ThyA is often used as a part of larger complexes of macromolecules that control replication and DNA repair.;Work with M. tuberculosis lysates indicated that ThyA and ThyX levels were relatively low in cells when compared to mammalian ThyA and expression levels remained constant between in replicating and non-replicating mycobacteria. These low target levels may provide an additional avenue for selective pathogen inhibition in the absence of enzyme specific inhibitors.;These studies were essential first steps in setting the groundwork to develop new antitubercular drugs targeting nucleotide metabolism in M. tuberculosis..