The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases.

3-甲基巴豆酰辅酶 A (CoA) 羧化酶 (MCC)、丙酮酸羧化酶和丙酰辅酶 A 羧化酶属于位于线粒体中的生物素依赖性羧化酶家族。它们参与人体的多种代谢途径，如氨基酸代谢和三羧酸循环。许多人类疾病都是由这些酶的突变引起的，但迄今为止它们的结构尚未完全解析。在这里，我们报告了一种优化的纯化策略，以获得不同构象状态的完整人内源性MCC、丙酰辅酶A羧化酶和丙酮酸羧化酶的高分辨率结构。我们还确定了与不同底物结合的 MCC 的结构。对不同状态MCC结构的分析揭示了底物诱导、多元素协同激活MCC的机制。这些结果为深入了解生物素依赖性羧化酶家族的催化机制提供了重要见解，对于开发治疗相关疾病的新药具有重要价值。