The biosynthesis of thymine (a DNA base) is essential in all organisms. The last step in this biosynthesis in humans and other eukaryotes is catalyzed by thyA/TYMS-encoded thymidylate synthase (TSase), and its cofactor is recycled by the folA-encoded dihydrofolate reductase (DHFR). In several human pathogens, e.g., those causing anthrax, tuberculosis, typhus, and more, the thyX-encoded flavin-dependent thymidylate synthase (FDTS) provides an alternative biosynthetic path to thymine. At first glance, FDTS seems merely to combine the activities of TSase and DHFR; it has same reactants and products as bi-functional TSase-DHFR. However, FDTS has very different genetic, structural, and mechanistic properties than its human counterparts. The catalytic mechanism of FDTSs is not understood; they have no known potent inhibitors; and inhibitors of classical TSases or DHFRs do not efficiently inhibit FDTSs. Were their mechanism known, rational inhibitor design could lead to new classes of antibiotic drugs with the potential for low toxicity. This proposal aims at studies of the chemical mechanism of FDTS catalysis. This study is of broader interest as preliminary studies suggested that FDTS chemical mechanism is different from that of either bifunctional TSase- DHFRs or any other known mechanism of nucleotide methylation. The proposed studies will employ a broad arsenal of methodologies, including isotopic labeling, single-turnover trapping of reaction intermediates, pre- steady-state and steady-state enzyme kinetics, time-resolved ESI-MS, mutagenesis, alternative cofactors, X-ray crystallography, and the synthesis and testing of putative intermediates. The findings from these diverse mechanistic studies present will test various proposed mechanisms and will illuminate the enigmatic mechanism of this enzyme.
Four specific aims are proposed:
Specific Aim 1 : Trapping and Identification of Intermediates.
Specific Aim 2 : Examination of the putative exocyclic methylene intermediate.
Specific Aim 3 : Structural studies.
Specific Aim 4 : Using 5-deaza-FADH2 as a mechanistic tool.

Public Health Relevance

The understanding of an enzyme's mechanism is fundamental to rational drug design. Mechanistic exploration of an understudied enzyme that represents a new path to DNA biosynthesis in several human pathogens is proposed. Findings will expand our scope of enzymatic mechanisms and will facilitate the development of new antibiotics of Biodefense and Public Health capabilities.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Barski, Oleg
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University of Iowa
Schools of Arts and Sciences
Iowa City
United States
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Mondal, Dibyendu; Koehn, Eric M; Yao, Jiajun et al. (2018) Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate. Bioorg Med Chem 26:2365-2371
Karunaratne, Kalani; Luedtke, Nicholas; Quinn, Daniel M et al. (2017) Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier. Arch Biochem Biophys 632:11-19
Mishanina, Tatiana V; Yu, Liping; Karunaratne, Kalani et al. (2016) An unprecedented mechanism of nucleotide methylation in organisms containing thyX. Science 351:507-10
Choi, Michael; Karunaratne, Kalani; Kohen, Amnon (2016) Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target. Molecules 21:
Mishanina, Tatiana V; Kohen, Amnon (2015) Synthesis and application of isotopically labeled flavin nucleotides. J Labelled Comp Radiopharm 58:370-5