Structural biology of biotin-dependent carboxylases

描述（由申请人提供）：生物素依赖性羧化酶使用共价附着的生物素辅助因子作为羧基生物素运输二氧化碳。四种人类生物素依赖性羧化酶是大型多酶复合物，在奇链脂肪酸氧化、支链氨基酸分解代谢、脂肪酸合成和糖异生等代谢途径中发挥核心作用。三种人类生物素依赖性羧化酶基因的突变与酶缺乏以及由此导致的代谢和发育障碍有关：丙酸血症、3-甲基丁基糖醛酸血症和乳酸血症。这些酶的结构-功能研究对于理解组装和催化机制非常有价值，通过研究这些多酶复合物的组织，对酶功能重要的活性位点特征和残基，以及在缺陷患者中发现的错义突变可能产生的结构和功能后果。由于其相对容易分离和可用于体外研究，从丙酸细菌中提取的转羧化酶多酶复合物长期以来一直被作为人类生物素依赖性羧化酶的模型系统。本应用程序主要研究细菌转羧化酶的结构和功能，以及两种人类酶，丙酰辅酶a羧化酶和甲基戊酰辅酶a羧化酶。广泛的目标是开展这些酶的结构-功能研究，以便更好地了解它们作为多酶复合物的组装及其催化活性机制。具体目标是：1。采用X射线晶体学和诱变相结合的方法研究生物素转羧化酶12S和5S亚基的羧化和脱羧机制。2. 采用生物物理与生物化学相结合的方法，探讨活性位点赖氨酸氨基甲酰化在转羧化酶5S亚基催化的羧化转移酶反应机制中的相关性。3. 研究转羧化酶的多亚基形式和人用酶丙酰辅酶a羧化酶和甲基戊酰辅酶a羧化酶的高分辨率结构。这些结构将为亚基-亚基界面和人类酶的细节提供前所未有的视角，但也将作为更准确的模板，用于人类酸血症突变可能的分子后果的计算建模。生物素依赖性酶在人体代谢中起重要作用。在患有代谢和发育障碍的患者中发现了改变基因的突变。研究这些酶的结构-功能关系将有助于理解它们如何组装和起作用，以及突变如何导致疾病。

DESCRIPTION (provided by applicant): Biotin-dependent carboxylases use a covalently attached biotin cofactor to transport carbon dioxide as carboxybiotin. The four human biotin-dependent carboxylases are large multi-enzyme complexes that play central roles in metabolic pathways such as oxidation of odd-chain fatty acids, catabolism of branched amino acids, fatty acid synthesis, and gluconeogenesis. Mutations in three of the human biotin-dependent carboxylase genes are associated with enzyme deficiencies and the resulting metabolic and developmental disorders propionic acidemia, 3-methylcrotonylglycinuria, and lactic acidemia. Structure-function studies of these enzymes are very valuable in understanding mechanisms of assembly and catalysis, by investigating the organization of these multi-enzyme complexes, the active site features and residues important for enzyme function, and the possible structural and functional consequences of missense mutations identified in deficiency patients. Due to its relative ease of isolation and availability for in vitro studies, the transcarboxylase multi-enzyme complex from propionic acid bacteria has long served as a model system for the human biotin- dependent carboxylases. This application focuses on structure-function studies of the bacterial transcarboxylase and on two human enzymes, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The broad objective is to carry out structure-function studies of these enzymes in order to better understand their assembly as multi-enzyme complexes and their mechanisms of catalytic activity. The specific aims are: 1. To investigate the mechanisms of biotin carboxylation and decarboxylation in the transcarboxylase 12S and 5S subunits respectively, using a combination of X- ray crystallography and mutagenesis. 2. To probe the relevance of active site lysine carbamylation in the mechanism of the carboxyltransferase reaction catalyzed by the transcarboxylase 5S subunit using a combination of biophysical and biochemical methods. 3. To pursue high resolution structures of multi-subunit forms of transcarboxylase and of the human enzymes propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. These structures will provide unprecedented views of subunit- subunit interfaces and details of the human enzymes, but will also serve as more accurate templates for the computational modeling of the possible molecular consequences of human acidemia mutations.Biotin-dependent enzymes are important in human metabolism. Mutations which alter their genes are found in patients with metabolic and developmental disorders. Investigating the structure-function relationships of these enzymes will aid understanding of how they assemble and function, and of how mutations may cause disease.