PMID:24422556

Citation	Becker, HF, Djaout, K, Lamarre, I, Ulmer, JE, Schaming, D, Balland, V, Liebl, U, Myllykallio, H and Vos, MH (2014) Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX. Biochem. J. 459:37-45
Abstract	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 re-oxidize 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 how the 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 micro-organisms.
Links	PubMed Online version:10.1042/BJ20131567
Keywords	Animals; Bacterial Proteins/metabolism; Binding Sites/physiology; Catalytic Domain/physiology; Cattle; Molecular Dynamics Simulation; Oxygen Consumption/physiology; Protein Binding/physiology; Substrate Specificity/physiology; Thymidylate Synthase/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:A0A0K2D0F3		GO:0032259: methylation	ECO:0000315:		P		Figure 1 showed that ThyX proteins also act as NADPH oxidase by reacting directly with O2.	complete CACAO 11584

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