Structural biology of biotin-dependent carboxylases

生物素依赖性羧化酶的结构生物学

• 批准号: 8009397
• 负责人: VIVIEN YEE
• 金额: $ 30.78万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009397
• 关键词: Acidity; Active Sites; Address; Affect; Amino Acid Substitution; Amino Acids; Bacteria; Binding; Biochemical; Biological Models; Biotin; Carbon Dioxide; Carboxyltransferases; Catabolism; Catalysis; Catalytic Domain; Cobalt; Complex; Computer Simulation; Critiques; Crystallization; Data; Decarboxylation; Dependence; Disease; Enzymes; Fatty Acids; Genes; Gluconeogenesis; Human; Image; In Vitro; Ions; Label; Lead; Ligand Binding; Ligands; Lysine; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; Methylmalonyl-CoA carboxyltransferase; Missense Mutation; Molecular; Monitor; Multienzyme Complexes; Mutagenesis; Mutation; N1' -carboxybiotin; Nature; Outcome; Oxaloacetates; Patients; Play; Population; Preparation; Principal Investigator; Propionic Acids; Proteins; Protons; Publishing; Pyruvate; Raman Spectrum Analysis; Reaction; Relative (related person); Research Design; Research Methodology; Resolution; Role; Sampling; Shapes; Site-Directed Mutagenesis; Spectrum Analysis; Staining method; Stains; Stretching; Structure; Structure-Activity Relationship; Techniques; Testing; X-Ray Crystallography; Yeasts; carbon dioxide transport; carboxylation; cofactor; deprotonation; design; developmental disease; enolate; enzyme deficiency; inhibitor/antagonist; ketotic hyperglycinemia; lactic acidemia; methylmalonyl-CoA decarboxylase; novel; oxidation; particle; programs; propionyl-coenzyme A; protein folding; research study; response; structural biology; tiglyl-coenzyme A; tool

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.

描述（由申请方提供）：生物素依赖性羧化酶使用共价连接的生物素辅因子作为羧基生物素转运二氧化碳。四种人类生物素依赖性羧化酶是大型多酶复合物，在代谢途径如奇数链脂肪酸的氧化、支链氨基酸的催化、脂肪酸合成和脂肪酸异生中发挥中心作用。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产生的代谢和发育障碍丙酸血症、3-甲基巴豆酰甘氨酸尿症和乳酸血症相关。这些酶的结构-功能研究是非常有价值的了解组装和催化的机制，通过调查这些多酶复合物的组织，活性位点的功能和残基重要的酶的功能，以及可能的结构和功能的错义突变缺陷患者中确定的后果。由于其相对容易分离和可用于体外研究，来自丙酸细菌的转羧酶多酶复合物长期以来一直用作人生物素依赖性羧化酶的模型系统。本申请集中于细菌转羧酶的结构-功能研究和两种人类酶，丙酰辅酶A羧化酶和甲基巴豆酰辅酶A羧化酶。广泛的目标是进行这些酶的结构-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的机制。具体目标是：1.利用X射线晶体学和诱变相结合的方法，研究转羧酶12 S和5S亚基生物素羧化和脱羧的机理。2.结合生物物理和生物化学方法探讨转羧酶5S亚基催化的羧基转移酶反应机制中活性位点赖氨酸氨甲酰化的相关性。3.追求高分辨率结构的多亚基形式的转羧酶和人类酶丙酰辅酶A羧化酶和甲基巴豆酰辅酶A羧化酶。这些结构将提供前所未有的亚基-亚基界面和人类酶的细节，但也将作为更准确的模板，用于人类酸血症突变的可能分子后果的计算建模。生物素依赖性酶在人类代谢中很重要。在患有代谢和发育障碍的患者中发现了改变其基因的突变。研究这些酶的结构-功能关系将有助于了解它们如何组装和发挥功能，以及突变如何导致疾病。