Structural Plasticity of Flavin-Dependent Thymidylate Synthase Controlled by the Enzyme Redox State

2'-Deoxythymidine-5'-monophosphate, dTMP, is an essential precursor of thymine, one of the four canonical bases of DNA. In almost all living organisms, dTMP is synthesized de novo by a reductive methylation reaction of 2'-deoxyuridine-5'-monophosphate (dUMP) catalyzed by the thymidylate synthase, where the carbon used for the methylation is derived from methylenetetrahydrofolate (CH2THF). Many microbes, including human pathogens, utilize the flavin-dependent thymidylate synthase encoded by the <i>thyX</i> gene to generate dTMP. The mechanism of action relies on the reduced coenzyme FADH^-, which acts both as a mediator, facilitating methylene transfer from CH2THF to dUMP, and as a reducing agent. Here, we present for the first-time crystallographic structures of ThyX from <i>Thermotoga maritima</i> in the reduced state alone and in complex with dUMP. ThyX flavin reduction appears to order the active site, favoring a flavin conformation that drastically deviates from that observed in the oxidized enzyme. The structures show that FADH^- potentially controls access to the folate site and the conformation of two active site loops, affecting the degree of accessibility of substrate pockets to the solvent. Our results provide the molecular basis for the sequential enzyme mechanism implemented by ThyX during dTMP biosynthesis.