Abstract
Background Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a 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. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.Methodology/Principal FindingsAfter cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k cat values. One possible explanation for the low catalytic activity of M. tuberculosis 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, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Conclusions/SignificanceThus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis.
Highlights
An estimated 2 million deaths occur annually due to infections of Mycobacterium tuberculosis, the causative agent of tuberculosis [1]
It has recently been postulated that paminosalicylic acid, a commonly used antitubercular drug, involves M. tuberculosis thymidylate synthase (ThyA), suggesting that inhibition of ThyA activity may be detrimental to M. tuberculosis growth and survival [12]
We report on the expression, purification, kinetic properties, and inhibitor-binding preferences of both M. tuberculosis ThyA and ThyX
Summary
An estimated 2 million deaths occur annually due to infections of Mycobacterium tuberculosis, the causative agent of tuberculosis [1]. Many established and experimental anticancer drugs, such as 5-fluorouracil, Nolatrexed (AG337), Pemetrexed, and Raltitrexed (ZD1694), act through this enzyme [10] Prior understanding of this general target in pharmacology, along with the vast number of potent lead inhibitors already available, should facilitate drug development against other indications including antibacterials [11]. The M. tuberculosis genome sequence lacks thymidine kinase, underscoring the essentiality of de novo TMP synthesis in this pathogen [13]. On this basis, selective inhibition of M. tuberculosis growth by ThyA inhibitors might not even require inhibitors selective for M. tuberculosis ThyA, as shown for the experimental treatment of malaria by inhibition of ThyA [14,15]. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary
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