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.
Dissertation
Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2018
DOI: 10.17077/etd.47m7o9mn
Abstract
Details
- Title: Subtitle
- Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase
- Creators
- Kalani Udara Karunaratne - University of Iowa
- Contributors
- Amnon Kohen (Advisor)Daniel M. Quinn (Advisor)Mishtu Dey (Committee Member)F. Christopher Pigge (Committee Member)James B. Gloer (Committee Member)Ashley Spies (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Chemistry
- Date degree season
- Summer 2018
- DOI
- 10.17077/etd.47m7o9mn
- Publisher
- University of Iowa
- Number of pages
- xv, 94 pages
- Copyright
- Copyright © 2018 Kalani Udara Karunaratne
- Language
- English
- Date submitted
- 11/19/2018
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 87-94).
- Public Abstract (ETD)
Antibiotics have been in use to treat bacterial infections for more than 80 years. Over-prescription and misuse of these antibiotics along with fast evolution of bacteria have reduced the effectiveness of these antibiotics. With time, pathogenic bacteria have adapted and developed resistance towards antibiotics. With the rise of antibiotic resistance, the demand to identify new targets for development of antibiotics has increased. The recently discovered enzyme Flavin-Dependent Thymidylate Synthase (FDTS) is one such target. FDTS is solely responsible for the thymidylate synthesis – a key cellular process, as this compound enters the DNA synthesis stream – in pathogenic bacteria such as B. anthracis, H. pylori and Rickettsia species. Interestingly, this enzyme is absent in humans, and the thymidylate synthase found in humans has no mechanistic or structural similarities with FDTS. This flavoprotein catalyzes the reductive methylation of uridylate (2'-deoxyuridine-5'-monophosphate; dUMP) to furnish thymidylate (2'-deoxythymidine-5'-monophosphate; dTMP). The carbon source for the reductive methylation is N5,N10-methylene-5,6,7,8 tetrahydrofolate (CH2H4fol), and the reduced flavin prosthetic group provides the reducing hydride to form the methyl group on C5 of dTMP. Despite the efforts towards understanding the mechanism of FDTS catalysis for the past two decades, detailed understanding of the mechanistic intricacies has not been realized yet. Compounds that mimic intermediates and transition-state analogues are likely to bind FDTS tightly. Therefore, understanding the mechanism of FDTS is crucial for the rational drug design against FDTS, the outcomes of which have lower toxicity to humans. We utilized the rapid quench flow technique in heavy water to follow the hydrogen transfers in FDTS. This method allows for rapid mixing and termination of the reaction during the various stages of the reaction cycle between a few milliseconds and a few minutes and allows for insight that other approaches may miss. With this technique, we were able to identify the steps that determine the rate of the oxidative half-reaction of FDTS. Other approaches – including substitution of the natural flavin for its analogues with altered reactivity – made strides toward delineating the way in which this auxiliary molecule participates in the reaction. The progress described here may have value in the development of novel therapeutics.
- Academic Unit
- Chemistry
- Record Identifier
- 9983777166402771
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