Thymidylate (dTMP, one of the four building blocks of DNA) is synthesized by methylation of Uridylate (2’-deoxyuridine 5’-monophosphate) at the C5 position. In most eukaryotes, including humans, the enzyme thymidylate synthase (TSase) encoded by thyA gene catalyzes the reductive methylation of dUMP to form dTMP. However, a novel gene thyX, coding for a new class of TSase was discovered . This new class of TSases named as flavin-dependent thymidylate synthases (FDTSs) makes use of a noncovalently bound flavin adenine dinucleotide (FAD). Several disease-causing bacteria such as Rickettsia prowazekii (typhus-causing), Mycobacterium tuberculosis, and Bacillus anthracis rely on FDTS. Classical TSase and FDTS are substantially different in structure and chemical mechanism . Hence, understanding the FDTS mechanism may allow the development of mechanism-based antibiotics with minimal toxicity to humans. In this work we have utilized solvent isotope effects to probe the mechanism of this unique enzyme. Both quench flow and stopped flow experiments were carried out in H2O and in D2O. Rate of product formation and substrate consumption followed by quench flow experiments done in isotopic solvents were compared to calculate the solvent isotope effect. Stopped flow experiments in two solvents reveal the oxidation state of Flavin with respect to reaction time. Information obtained by these techniques will provide insight into the complex mechanism of FDTS catalyzed thymidylate synthesis.

Mechanistic studies in Flavin Dependent Thymidylate  Synthase (FDTS)

253rd National Meeting (2017)

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