α酮戊二酸脱氢酶复合体（OGDHc）是三羧酸循环中的关键限速酶和调控酶，其负责催化三羧酸循环中的α酮戊二酸经过氧化脱羧，生成琥珀酰辅酶A的同时产生NADH并释放CO2的反应。三羧酸循环在细胞代谢中处于枢纽地位，因此OGDHc关联多种代谢通路。OGDHc活性降低与癌症以及多种神经退行性疾病，如帕金森病（PD）和阿尔茨海默病（AD）有关。OGDHc是分子量约为4MDa的超大多酶分子机器，其由三种不同的酶组成。它们分别是α酮戊二酸脱氢酶（E1o），二氢硫辛酰胺琥珀酰转移酶（E2o）以及二氢硫辛酰胺脱氢酶（E3）。但是到目前为止，OGDHc的整体结构依然未知，且没有系统的方法对之进行研究。同时OGDHc在全酶组分中的各亚基的比例也争议较大。由于OGDHc在行使功能时需要各个组分之间配合，因此研究清楚全酶的结构和比例对于了解其功能具有重要意义。在本文的研究中，我们首先采用冷冻电镜单颗粒分析技术去解析得到了近原子分辨率的OGDHc的核心亚基3.3?的结构。同时单颗粒分析也进一步证实OGDHc整体结构的不均一性。更重要的是，我们通过冷冻电子断层成像结合子断层平均的方法，获得了7.9?的核心亚基E2o的结构，9.7?的外圈亚基E1o的结构以及12.2?的外圈亚基E3的结构。我们通过将获得的各亚基的结构进行重投射，重构出2014个完整的OGDHc的全酶结构。我们依据重构出的整体结构对于OGDHc全酶进行统计分析，确定了OGDHc中各亚基之间的数目和比例，以及外圈亚基在内核周围的分布规律。此外，我们通过生物物理和生物化学的方法，确定各亚基中组装元件之间的相互作用关系，并提出了完整的OGDHc组装模型。总体来说，我们的实验结果首次揭示了OGDHc的整体结构，通过结构生物学最直观的手段，确定了OGDHc中的各亚基之间的个数，比例以及外圈亚基在内核周围的分布规律，同时首次提出了完整哺乳动物OGDHc的组装模型。OGDHc整体结构的解析能够为基于OGDHc的功能研究，分子动力学模拟，靶向OGDHc的药物作用机制的研究以及药物研发提供了重要基础。
The 2-oxoglutarate dehydrogenase complex （OGDHc） is a critical rate-limiting and regulatory enzyme in the tricarboxylic acid （TCA） cycle. It catalyzes the oxidative decarboxylation of 2-oxoglutarate in the TCA cycle to generate succinyl-CoA, while producing NADH and releasing CO2. Given the central role of the TCA cycle in cellular energy and metabolic homeostasis, OGDHc is interconnected with multiple metabolic pathways. Reduced OGDHc activity has been linked to cancer and neurodegenerative disorders, including Parkinson’s disease （PD） and Alzheimer’s disease （AD）. OGDHc is a large multi-enzyme molecular machine with a molecular weight of approximately 4 MDa, composed of three distinct enzymes: 2-oxoglutarate dehydrogenase （E1o）, dihydrolipoamide succinyltransferase （E2o）, and dihydrolipoamide dehydrogenase （E3）. However, the overall architecture of OGDHc remains unresolved, and systematic methodologies for its study are lacking. Furthermore, the stoichiometric ratios of its subunits within the holoenzyme remain highly debated. Since the functional integrity of OGDHc relies on precise coordination among its components, elucidating its structural organization and subunit composition is essential for understanding its mechanism. In this study, we employed cryo-EM single-particle analysis to resolve the structure of the core E2o subunit at 3.3 ?. This analysis also confirmed the structural heterogeneity of OGDHc. Moreover, using cryo-electron tomography combined with subtomogram averaging, we obtained 7.9 ?, 9.7 ?, and 12.2 ? resolution structures for the core E2o, peripheral E1o, and peripheral E3 subunits, respectively. By reprojecting these subunit structures, we reconstructed 2,014 intact OGDHc holoenzyme architectures. Statistical analysis of the subunit based on these reconstructions revealed the number and the stoichiometric ratios of subunits and the spatial distribution of peripheral subunits around the core. Additionally, through biophysical and biochemical approaches, we detected the interaction networks among constituent domains and proposed a comprehensive assembly model for OGDHc holoenzyme. In summary, our findings provide the first holistic structural characterization of OGDHc. Using direct structural biology approaches, we determined the stoichiometry, spatial distribution of peripheral subunits around the core, and proposed the first assembly model for the mammalian OGDHc. The resolved architecture of OGDHc establishes a critical foundation for functional studies, molecular dynamics simulations, and drug development targeting OGDHc.
