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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：
8008942
负责人：
VIVIEN YEE
金额：
$ 6.2万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-15 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8008942
关键词：
Acidity Active Sites Address Affect Amino Acid Substitution Amino Acids Animal Welfare Bacteria Bibliography Binding Biochemical Biological Models Biotin Carbon Dioxide Carboxyltransferases Catabolism Catalysis Catalytic Domain Cobalt Complex Country Critiques Crystallization Data Decarboxylation Dependence Disease Environment Environmental Impact Enzymes Equipment Fatty Acids Genes Gluconeogenesis Human IACUC Image In Vitro International Ions Label Lead Ligand Binding Ligands Metabolic Metabolic Diseases Metabolic Pathway Methods Methylmalonyl-CoA carboxyltransferase Missense Mutation Monitor Multienzyme Complexes Mutation N1&apos -carboxybiotin Nature Outcome Oxaloacetates Patients Play Population Preparation Principal Investigator Propionic Acids Proteins Protons Publishing Pyruvate Pyruvates Raman Spectrum Analysis Reaction Relative (related person)Research Research Design Research Ethics Committees Resolution Resources Role Sampling Shapes Site-Directed Mutagenesis Spectrum Analysis Staining method Stains Stretching Structure Techniques Testing Vertebrates Yeasts abstracting biotin 2 carbon dioxide transport carboxylation cofactor deprotonation design developmental disease enolate enzyme deficiency expiration human subject 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

Biotin-dependent carboxylases use a covalently attached biotin cofactor to transport carbon dioxide as carboxybiotin. Four human biotin-dependent carboxylases have been identified, and play central roles in metabolic pathways such as oxidation of odd-chain fatty acids, catabolism of branched amino acids, fatty acid synthesis, and gluconeogenesis. The bacterial transcarboxylase enzyme has long served as a model system for the human biotindependent carboxylases. This proposal focuses on structure-function studies of transcarboxylase and on two human enzymes, propionyl coenzyme A carboxylase and methylcrotonoyl coenzyme A carboxylase. The broad objective is to provide structural information for these enzymes in order to better understand their assembly as multi-enzyme complexes and their mechanisms of catalytic activity. The specific aims are to: 1. To probe the chemistry and mechanism of the transcarboxylase active sites with structural studies of the isolated 12S and 5S subunits. Crystals of the isolated subunits bound to substrate, product, or cofactor will be studied by X-ray crystallography and Raman crystallography. Site- directed mutagenesis will be used to probe the importance of residues highlighted by structural studies. 2. To probe the molecular architecture of the intact transcarboxylase multienzyme complex by X-ray crystallography and electron microscopy. 3. To investigate the crystal structure of human propionyl coenzyme A carboxylase and to model the possible structural consequences of deficiency mutations in the metabolic disorder propionic acidemia. 4. To investigate the crystal structure of the human methylcrotonyl coenzyme A carboxylase beta subunit and to model the possible structural consequences of deficiency patients with the metabolic disorder 3-methylcrotonylglycinuria. Relevance: Biotin-dependent enzymes are important in human metabolism. Mutations which alter their genes are found in patients with metabolic and developmental disorders. Investigating the structures of these enzymes will aid understanding of how these proteins assemble and function, and help explain how mutations may cause disease.

生物素依赖性羧化酶使用共价连接的生物素辅因子将二氧化碳作为羧基生物素运输。四种人类生物素依赖性羧化酶已被鉴定，并且在代谢途径中发挥核心作用，例如奇数链脂肪酸的氧化、支链氨基酸的分解代谢、脂肪酸合成和糖异生。细菌转羧酶长期以来一直作为人类生物素依赖性羧化酶的模型系统。该提案重点关注转羧酶和两种人类酶（丙酰辅酶 A 羧化酶和甲基巴豆酰辅酶 A）的结构功能研究羧化酶。广泛的目标是提供这些酶的结构信息，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. 探讨转羧酶的化学性质和作用机制活性位点以及分离的 12S 和 5S 亚基的结构研究。与底物、产物或辅因子结合的分离亚基的晶体将通过 X 射线晶体学和拉曼晶体学进行研究。定点诱变将用于探讨结构研究强调的残基的重要性。 2. 通过X射线晶体学和电子显微镜探测完整转羧酶多酶复合物的分子结构。 3. 研究人丙酰辅酶 A 羧化酶的晶体结构，并模拟代谢紊乱丙酸血症中缺陷突变可能产生的结构后果。 4. 研究晶体结构人类甲基巴豆酰辅酶 A 羧化酶 β 亚基的研究，并模拟患有代谢性疾病 3-甲基巴豆酰甘氨酸尿症的缺陷患者可能的结构后果。相关性：生物素依赖性酶在人体新陈代谢中很重要。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。研究这些酶的结构将有助于了解这些蛋白质如何组装和发挥作用，并有助于解释突变如何导致疾病。