The Oxidase Reaction of Flavin-Dependent Thymidylate Synthase Reveals the Nature of Inhibitor Binding

Escalating antimicrobial resistance presents an urgent need for novel antibiotics, particularly against Mycobacterium tuberculosis ( M. tb ), which claims 1.5 million lives annually. A promising avenue for antibiotic development is inhibiting DNA biosynthesis by targeting Thymidylate Synthase enzymes (TS) that produce 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA. Two forms of TS enzymes are known, and M. tb has been shown to rely on a unique flavin-dependent thymidylate synthase (FDTS), which is distinct from the human TSase enzyme. This work explores the mechanism and binding modes of inhibitors of the Mtb FDTS enzyme by utilizing a nonproductive oxidase reaction (O 2 reduction to H 2 O 2 ) catalyzed by the flavin cofactor. We discovered that inhibitors of Mtb FDTS that bind competitively at the nucleotide-binding site potentiate the oxidase reaction. Conversely, inhibitors of the enzyme that bind at the folate's binding site abate the oxidase reaction. We exploit this contrasting kinetic behavior to show that naphthoquinones inhibit FDTS by binding competitively at the nucleotide's active site, activating the enzyme's oxidase reaction and consequently producing substantial peroxide as a byproduct. We confirm our predicted inhibitor binding modes through direct measurement of folinic acid binding to the ternary enzyme-flavin-naphthoquinone complex, providing a revised kinetic mechanism for the FDTS-catalyzed reaction. Our findings show that naphthoquinones inhibit the native FDTS reaction and divert the enzyme's activity to produce reduced oxygen species, which sheds light on their antimycobacterial activity and will be critical for future inhibitor development and high-throughput screening (HTS) methods.