Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany; Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3, Halle/Saale, Germany.
Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, Halle/Saale, Germany.
Cell Rep. 2021 Feb 9;34(6):108727. doi: 10.1016/j.celrep.2021.108727.
The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.
丙酮酸脱氢酶复合物（PDHc）是一种参与丙酮酸氧化的巨大酶组装体。PDHc 组件已经在分离状态下进行了表征，但由于其巨大的尺寸、异质性和可变性，复合物的四级结构仍然难以捉摸。在这里，我们在天然细胞提取物中鉴定出完全组装的嗜热毛壳菌α-酮酸脱氢酶复合物，并利用质谱、活性测定、交联、电子显微镜（EM）和计算建模来表征其结构域排列。我们报告了 PDHc 核心的冷冻电镜结构，并观察到了以前未知的天然状态的独特特征。10-MDa PDHc 的非对称重建解决了其组件的空间接近性，与计量数据（60 E2p：12 E3BP：∼20 E1p：≤12 E3）一致，并提出了组件酶之间的最小反应途径。PDHc 显示存在一个动态的丙酮酸氧化隔室，由核心和外周蛋白种类组织。我们的数据为进一步理解 PDHc 和α-酮酸脱氢酶复合物的结构和功能提供了一个框架。
