Pecqueur Ludovic, Lombard Murielle, Hamdane Djemel
Laboratoire de Chimie des Processus Biologiques, CNRS-UMR 8229, Collège De France, Université Pierre et Marie Curie, 11 place Marcelin Berthelot, CEDEX 05, 75231 Paris, France.
Biomolecules. 2025 Feb 21;15(3):318. doi: 10.3390/biom15030318.
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 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 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.
2'-脱氧胸苷-5'-单磷酸（dTMP）是胸腺嘧啶的重要前体，胸腺嘧啶是DNA的四种标准碱基之一。在几乎所有的生物体中，dTMP是由胸苷酸合成酶催化2'-脱氧尿苷-5'-单磷酸（dUMP）的还原甲基化反应从头合成的，其中用于甲基化的碳来自亚甲基四氢叶酸（CH2THF）。许多微生物，包括人类病原体，利用由该基因编码的黄素依赖性胸苷酸合成酶来生成dTMP。其作用机制依赖于还原型辅酶FADH，它既作为介质促进亚甲基从CH2THF转移到dUMP，又作为还原剂。在这里，我们首次展示了来自[具体来源未提及]的ThyX单独处于还原状态以及与dUMP形成复合物时的晶体结构。ThyX黄素还原似乎使活性位点有序化，有利于一种与氧化态酶中观察到的构象有很大偏差的黄素构象。这些结构表明FADH可能控制对叶酸位点的 access以及两个活性位点环的构象，影响底物口袋对溶剂的可及程度。我们的结果为ThyX在dTMP生物合成过程中实施的顺序酶机制提供了分子基础。
