Structural biology of biotin-dependent carboxylases

生物素依赖性羧化酶的结构生物学

基本信息

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
项目类别：
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&apos -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

项目摘要

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（由申请人提供）：依赖生物素的羧化酶使用共价附着的生物素辅因子将二氧化碳作为羧蛋白运输。四种人生物素依赖性羧化酶是大型多酶复合物，它们在代谢途径中起着核心作用，例如奇怪的链脂肪酸的氧化，分支氨基酸的分解代谢，脂肪酸合成和糖生成。人类生物素依赖性羧化酶基因的突变与酶缺乏症以及由此产生的代谢和发育障碍丙酸血症，3-甲基氯酸糖酸糖尿病和乳酸血症有关。这些酶的结构功能研究对于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的组织，以及在缺乏症患者中鉴定出的错义突变的可能的结构和功能后果，对理解组装和催化的机制非常有价值。由于其相对易于分离和体外研究的可用性，因此从丙酸细菌的经羧化酶多酶复合物长期以来一直是人类依赖生物素的羧化酶的模型系统。该应用的重点是细菌转羧化酶的结构功能研究以及两种人类酶，丙二酰辅酶A羧化酶和甲基氯苯甲酰-COA羧化酶。广泛的目标是对这些酶进行结构功能研究，以便更好地理解它们作为多酶复合物及其催化活性机制。具体目的是：1。分别使用X-Ray晶体学和诱变的组合研究了经羧化酶12S和5S亚基中生物素羧化和脱羧的机制。 2。为了探测活性位点赖氨酸氨基氨基化在羧基转移酶反应的机理中，使用生物物理和生化方法的组合通过转羧化酶5S亚基催化。 3。追求多生成形式的转羧酸酶和人酶的高分辨率结构，丙酰辅酶A羧化酶和甲基氯酸碳酸二氧基羧化酶。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观点，但也将作为更准确的模板，用于对人类酸血症突变的可能分子后果的计算建模。Biotin依赖性酶在人类代谢中很重要。改变其基因的突变是在代谢和发育障碍患者中发现的。研究这些酶的结构 - 功能关系将有助于理解它们的组装和功能以及突变可能导致疾病的方式。