Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX

Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2′-deoxythymidine-5′-monophosphate) from dUMP (2′-deoxyuridine-5′-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O₂. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O₂-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ∼30 s ^-1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O₂ requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ∼3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH₂H₄folate (N⁵,N¹⁰- methylene-5,6,7,8-tetrahydrofolate) was found to reoxidize FADH₂ within 1 ms, thus very efficiently competing with FADH₂ oxidation by O₂ (1.5 s^-1 under aerobic conditions). The resulting reaction scheme points out howthe interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic microorganisms.