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 anti-microbial target. It binds FAD and three substrates, producing deoxythymidine-5’-monophosphate from deoxyuridine-5’-monophosphate (dUMP). However, ThyX proteins also act as NADPH oxidase by directly reacting with oxygen. 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 oxygen-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 oxygen 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 CH2H4 folate 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, while not rate-limiting 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.

Source: BJ Energy - January 15, 2014 Category: Biochemistry Authors: H F. Becker, K Djaout, I Lamarre, J E. Ulmer, D Schaming, V Balland, U Liebl, H Myllykallio, M H. Vos Tags: BJ Biomolecules Source Type: research