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Abstract：
Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
摘要：
亚甲基四氢叶酸还原酶（MTHFR）是连接叶酸和蛋氨酸甲基循环的关键黄素蛋白，催化亚甲基四氢叶酸转化为甲基四氢叶酸。人类MTHFR（hMTHFR）经历了复杂的变构调节，涉及蛋白质磷酸化和S-腺苷甲硫氨酸（AdoMet）依赖性抑制，尽管由于缺乏功能结构模型，诸如亚基方向和FAD状态等其他因素仍未得到充分研究。这里,我们报道了活性（R）和抑制（T）状态下的嗜热ChaetomiumMTHFR（cMTHFR）的晶体结构。我们揭示了T状态下Tyr361的FAD闭塞，防止底物相互作用。值得注意的是,cMTHFR的抑制形式每个亚基容纳两个AdoMet分子。此外,我们对hMTHFR中的磷酸化位点进行了详细的研究，其中三个以前身份不明。基于我们的cMTHFR模型提供的结构框架，我们提出了一种可能的机制来解释MTHFR的变构结构转变，包括磷酸化对AdoMet依赖性抑制的影响。
