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Oxidase activity of a flavin‐dependent thymidylate synthase

Flavin‐dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N5,N10‐methylene‐5,6,7,8‐tetrahydrofolate (CH2H4folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O2 to H2O2, using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady‐state and single‐turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH2H4folate competitively inhibits the oxidase activity, which indicates that CH2H4folate and O2 compete for the same reduced and dUMP‐activated enzymatic complex (FDTS–FADH2–NADP+–dUMP). These studies imply that the binding of CH2H4folate precedes NADP+ release during FDTS activity. The inhibition constant of CH2H4folate towards the oxidase activity was determined to be rather small (2 μm), which indicates a tight binding of CH2H4folate to the FDTS–FADH2–NADP+–dUMP complex.