In humans and most eukaryotes, thymidylate synthase (TSase) serves as a key enzyme that catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to synthesize deoxythymidine monophosphate (dTMP), a key component of DNA. The N5, N10- methylene-5,6,7,8-tetrahydrofolate (MTHF) serves as both the methylene donor and the hydride donor while generating dihydrofolate (H2folate) as the byproduct. However, in 2002, Myllykallio reported the discovery of flavin-dependent thymidylate synthase (FDTS) that also functions to maintain the dTMP pool, although the mechanism is different. Since then, considerable progress was made in characterizing this enzyme. It was found that structurally FDTS is substantially different from TSase both with respect to structure and with respect to the mechanistic pathway of catalysis. In the FDTS-catalyzed methylation of dUMP, MTHF serves only as the methylene donor, generating tetrahydrofolate (H4folate), unlike TSase, and FDTS utilizes NADPH as a reductant. Activity of the enzyme depends on the presence of the noncovalently bound prosthetic group, flavin adenine dinucleotide (FAD). Interestingly, the enzyme FDTS is present in several human pathogens that cause diseases including syphilis, tuberculosis, anthrax poisoning, typhus, botulism, peptic ulcers and more, but is absent in humans; thus, it poses an attractive target for antibiotics. In the modern world, antibiotic resistance is a menace; consequently, new targets for new antibiotics are being sought. Hence, elucidating the chemical mechanism of FDTS is of paramount interest, as we and others believe this could allow for rational design of drugs that selectively target these pathogens with minimal human toxicity.
Although several chemical mechanisms for FDTS catalysis have been put forward, complete understanding has still not been achieved. One of the primary concerns was the role of FAD in catalysis, and we found – as described in Chapter II and III – that FAD is a methylene carrier rather than just a hydride donor, as previously postulated. Secondly, all mechanisms proposed so far predict the presence of a noncovalently bound putative exocyclic methylene intermediate (an isomer of dTMP) occurring in the catalytic pathway of FDTS. However, direct evidence to prove its existence was lacking. Recently, we have been able to synthesize this intermediate, as described in Chapter IV. As shown in Chapter V and VI, we used steady-state kinetics, isotopic substitution and NMR studies to test this intermediate with FDTS. We believe our findings will greatly improve the understanding of this enzyme and will impact drug design by government agencies, pharmaceutical companies, and academic laboratories.