Document Type


Date of Degree

Summer 2018

Access Restrictions

Access restricted until 08/31/2020

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Kohen, Amnon

Second Advisor

Quinn, Daniel M.

First Committee Member

Dey, Mishtu

Second Committee Member

Pigge, F. Christopher

Third Committee Member

Gloer, James B.

Fourth Committee Member

Spies, Ashley


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

Available for download on Monday, August 31, 2020

Included in

Chemistry Commons