Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX

• Published in: PloS one 2008-05, Vol.3 (5), p.e2237
• Main Authors: Hunter, Joshua H, Gujjar, Ramesh, Pang, Cullen K T, Rathod, Pradipsinh K
• Format: Article
• Language: English
• Subjects: Adenosine; Analysis; Antitubercular agents; Base Sequence; Biochemistry; Catalysis; Catalytic activity; Chemotherapy; Chromatography; Chromatography, High Pressure Liquid; Cloning; Dehydrogenases; Deoxyribonucleic acid; Dihydrofolate reductase; DNA; DNA biosynthesis; DNA Primers; DNA repair; Drug development; Drug resistance; Drugs; E coli; Enzyme Inhibitors - pharmacology; Enzymes; Flavin; Folic acid; Genetic aspects; Genomes; Genomics; High-performance liquid chromatography; Infectious Diseases; Inhibitors; Isoenzymes - metabolism; Kinases; Kinetics; Ligands; Liquid chromatography; Macromolecules; Malaria; Metabolites; Microbiology; Molecular weight; Mutagenesis; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Pathogens; Pharmaceutical sciences; Pharmacology; Plasmodium falciparum; Proteins; Reaction kinetics; Reaction products; Saturation; Substrate Specificity; Substrates; Target recognition; Thymidylate synthase; Thymidylate Synthase - antagonists & inhibitors; Thymidylate Synthase - isolation & purification; Thymidylate Synthase - metabolism; Titration; Tuberculosis; Turnover rate; Vitamin B
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0002237

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. After 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. Thus, 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.