Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD + -dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila . By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.

中文翻译：

---

无序到有序的活性位点封顶调节肌醇途径的限速步骤


1-磷酸肌醇合酶 (MIPS) 催化 6-磷酸葡萄糖 (G6P) 经 NAD + 依赖性异构化为 1-磷酸肌醇 (IMP)，控制肌醇途径的限速步骤。先前的结构研究侧重于详细的分子机制，忽略了驱动这种 240 kDa 同源四聚体复合物功能的大规模构象变化。在这项研究中，我们鉴定了嗜热真菌 Thermochaetoides thermophila 细胞提取物中的活性内源 MIPS。通过解析 2.48 Å (FSC = 0.143) 处的天然结构，我们揭示了一个完全填充的活性位点。利用 3D 变异性分析，我们发现了 MIPS 的构象状态，使我们能够直接可视化其催化中心的有序到无序的转变。 G6P 的无环中间体在三种构象状态中的两种中占据了活性位点，这表明高能中间体的静电稳定在催化机制中起着至关重要的作用。对所有具有已知结构的异构酶的检查揭示了其活性位点内二级结构的类似波动。基于这些发现，我们建立了一个构象选择模型，该模型控制底物结合并最终控制肌醇的可用性。特别是，MIPS 的基态表现出与底物结合无关的结构构型，这是在各种异构酶中观察到的模式。这些发现有助于理解 MIPS 基于结构的功能，为未来针对调控和潜在治疗应用的研究提供模板。