The reactions of flavin-dependent thymidylate synthase from <i>Thermotoga maritima</i>
For decades the biosynthesis of thymidylate, a vital DNA precursor, was thought to be catalyzed solely by ThyA (the “classic” thymidylate synthase), which was thought to be found in all organisms. Recently a structurally unrelated new class of thymidylate synthase, ThyX, was found in many bacterial organisms including many pathogenic species. Both enzymes catalyze the reductive methylation of dUMP (deoxyuridine monophosphate) using CH2THF as a carbon source to form dTMP (deoxythymidine monophosphate). However, the reductive mechanism is different between these two enzymes.
The “classic” thymidylate synthase uses CH2THF as both the reductant and as the source of carbon forming both dTMP and dihydrofolate. ThyX catalyzes the reductive methylation of dUMP using CH2THF as the carbon source, but requires a Flavin prosthetic group and reducing equivalents from NADPH to form dTMP and THF (tetrahydrofolate). This reaction can be divided into two reactions. In the first reaction, the reductive half-reaction, the enzyme is reduced by NADPH. In the second reaction, the oxidative half-reaction, the reduced enzyme reacts with both dUMP and CH2THF to form dTMP and tetrahydrofolate.
The mechanisms of both the reductive and oxidative half-reactions were studied in detail by transient kinetics. The effect of the substrates on both reactions suggests a preferred binding order. The substrate, dUMP, binds first followed by reduction of the enzyme by NADPH. The reduced enzyme•dUMP complex then reacts with CH2THF to form dTMP and THF. Further experiments on the oxidative half-reaction by stopped-flow spectroscopy and rapid reaction acid quenching have revealed the accumulation of two as yet unidentified intermediates prior to oxidation of the flavin and product formation. Studies of the oxidative half-reaction using site directed mutagenesis, pH dependence, dUMP, and FAD analogues have let to a proposed detailed mechanism for the synthesis of dTMP by ThyX from Thermotoga maritima. This mechanism is different than the classical mechanism of ThyA. Strikingly, the activation of dUMP by ThyX does not occur by Michael addition, but occurs through the polarization of dUMP by binding to a polarized active site. The difference in these mechanisms, as well as there being no structural or sequence similarity in the proteins, makes ThyX an attractive target for antibiotic treatment of infections by many pathogens.