Study of Kinetic Mechanism of Flavin‐Dependent Thymidylate Synthase from Thermotoga Maritima

Abstract

Thymidylate, an essential DNA nucleotide, is synthesized de novo by all organisms. The enzyme thymidylate synthase catalyses the last committed step of this de novo pathway, which is the reductive methylation of 2′‐deoxyuridine‐5′‐monophosphate (dUMP) to produce 2′‐deoxythymidine‐5′‐monophosphate (dTMP). Most organism including humans, rely on classical thymidylate synthase. Recently the identification of many bacteria and human pathogens lacking this enzyme led to the discovery of flavin‐dependent thymidylate synthase (FDTS) encoded by the thyX gene. The two classes of enzyme share no structural or sequence homology and while the mechanism of classical enzyme has been studied over decades and is well established, the mechanism of thyX is still not clear. Overall, thyX receives reducing equivalents from NADPH and uses its flavin prosthetic group for redox chemistry and methylenetetrahydrofolate (CH2H4folate) as the methylene donor to produce dTMP with the concomitant release of tetrahydrofolate. Many different mechanisms have been proposed for the oxidative half‐reaction of this enzyme. One of the major questions involving its catalytic mechanisms is the activation of the pyrimidine substrate. Here we tested the feasibility of a recently published mechanism by synthesizing one of the proposed intermediates. We also present kinetic and spectroscopic data that provides more details into the kinetic mechanism of this flavoenzyme.