Date of Degree
Access restricted until 08/31/2020
PhD (Doctor of Philosophy)
Daniel M. Quinn
First Committee Member
Second Committee Member
F. Christopher Pigge
Third Committee Member
James B. Gloer
Fourth Committee Member
All organisms must maintain an adequate level of thymidylate, which gets phosphorylated twice and then utilized by DNA polymerases for DNA replication that must precede cell division. Most organisms rely on classical thymidylate synthase (TSase) for this function. However, a subset of microorganisms – including a number of notable, widespread human pathogens – relies on an enzyme with a distinct structure and catalytic strategy. This enzyme is termed flavin-dependent thymidylate synthase (FDTS), as the flavin is required for thymidylate production. Because of this considerable orthogonality between FDTS and classical TSase, FDTS serves as a promising target for new therapeutics – one that could have only mild adverse effects on the host organism. FDTS catalyzes the reductive methylation of uridylate (2′-deoxyuridine-5′-monophosphate; dUMP) to yield thymidylate (2′-deoxythymidine-5′-monophosphate; dTMP). The methylene originally resides on CH2H4folate and is eventually transferred to the nucleotide. This methylene’s route to dUMP is unique in enzymology, and our experiments described herein strive to gain an understanding of the molecular details of its transfer. Compounds that mimic intermediates and transition states along this path are likely to bind FDTS tightly and could be leads for drugs, and our new insights could facilitate this. After methylene transfer is complete, a hydride transfer from flavin to the nucleotide occurs. We utilized rapid quench flow techniques in heavy water to follow the hydrogen transfers in FDTS; solvent isotope effects were measured and analyzed, furnishing evidence that the hydride transfer contributes to rate limitation. Reconstitution of the enzyme with unnatural flavins both reinforced these conclusions and suggested new hypotheses and experiments.
Enzymes, Flavin, Mechanisms, Thymidylate
xv, 94 pages
Includes bibliographical references (pages 87-94).
Copyright © 2018 Kalani Udara Karunaratne
Karunaratne, Kalani Udara. "Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase." PhD (Doctor of Philosophy) thesis, University of Iowa, 2018.
Available for download on Monday, August 31, 2020