Flavin-dependent thymidylate synthase: putting together the mechanistic puzzle from reaction intermediate pieces

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.