A better understanding of how enzymes activate covalent bonds will be pursued via the investigation of a small enzyme that catalyzes a single C-H bond activation, and these studies will be extended to a larger enzyme that catalyzes a complex cascade of covalent bond activations within a single active site. The studies aim to reveal the nature of the chemical step (bond activation), and the role of the whole protein structure and dynamics in that process. The studies will illuminate the evolutionary progressions that enhance the bond activation despite the fact that the catalytic turnover is usually rate-limited by processes other than the chemical transformation.
Four specific aims are proposed:
Aim 1 will follow the nature of the chemical step along the natural evolution of dihydrofolate reductase (DHFR) from bacteria to human, and from DHFR toward dihydrobiopterin reductase (DHPR) by means of directed evolution.
Aim 2 will examine the role of active site residues in different chemical conversions catalyzed by the enzyme thymidylate synthase (TSase), and will test experimentally an alternative reaction mechanism proposed by calculations.
Aim 3 will study the relations between the chemical step and fast equilibrium dynamics (femtosecond-nanosecond) across the whole protein.
Aim 4 will induce a minimal perturbation of those fast dynamics by means of isotopically heavy proteins (Born-Oppenheimer enzymes), and will explore the resultant effects on the catalyzed chemical step. Such comprehensive studies will require a broad arsenal of experimental and theoretical tools including measurements and calculations of kinetic isotope effects (KIEs);protein crystallography and measurements of anisotropic B-factors from X-ray diffractions;NMR relaxation measurements, hybrid QM/MM calculations;vibrational spectroscopy (2D-IR);and directed evolution. Accordingly, the research team is composed of fours subcontractors, three other co-investigators, and the PI.

Public Health Relevance

Both DHFR and TSase are targets for antibiotic and chemotherapeutic drugs. The proposed studies will expose otherwise hidden chemical steps thus providing new targets for inhibitors and drug design. Understanding of the evolution of new substrate specificity will reveal molecular and physical features in the evolution of drug resistance. The expected outcome of the proposed studies will also shed light on the question of whether protein dynamics should be considered in rational drug design and biomimetic catalyst design.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-A (02))
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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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Kholodar, Svetlana A; Ghosh, Ananda K; ?widerek, Katarzyna et al. (2018) Parallel reaction pathways and noncovalent intermediates in thymidylate synthase revealed by experimental and computational tools. Proc Natl Acad Sci U S A 115:10311-10314
?widerek, Katarzyna; Tuñón, Iñaki; Williams, Ian H et al. (2018) Insights on the Origin of Catalysis on Glycine N-Methyltransferase from Computational Modeling. J Am Chem Soc 140:4327-4334
?widerek, Katarzyna; Nödling, Alexander R; Tsai, Yu-Hsuan et al. (2018) Reaction Mechanism of Organocatalytic Michael Addition of Nitromethane to Cinnamaldehyde: A Case Study on Catalyst Regeneration and Solvent Effects. J Phys Chem A 122:451-459
?widerek, Katarzyna; Arafet, Kemel; Kohen, Amnon et al. (2017) Benchmarking Quantum Mechanics/Molecular Mechanics (QM/MM) Methods on the Thymidylate Synthase-Catalyzed Hydride Transfer. J Chem Theory Comput 13:1375-1388
Ranasinghe, Chethya; Guo, Qi; Sapienza, Paul J et al. (2017) Protein Mass Effects on Formate Dehydrogenase. J Am Chem Soc 139:17405-17413
Kholodar, Svetlana A; Kohen, Amnon (2016) Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction? J Am Chem Soc 138:8056-9
Guo, Qi; Gakhar, Lokesh; Wickersham, Kyle et al. (2016) Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii. Biochemistry 55:2760-71
Krzemi?ska, Agnieszka; Moliner, Vicent; ?widerek, Katarzyna (2016) Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease. J Am Chem Soc 138:16283-16298
Francis, Kevin; Sapienza, Paul J; Lee, Andrew L et al. (2016) The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase. Biochemistry 55:1100-6
?widerek, Katarzyna; Moliner, Vicent (2016) Computational Studies of Candida Antarctica Lipase B to Test Its Capability as a Starting Point To Redesign New Diels-Alderases. J Phys Chem B 120:2053-70

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