Structural biology of biotin-dependent carboxylases

描述(申请人提供)：生物素依赖的羧基酶使用共价连接的生物素辅因子以羧基生物素的形式运输二氧化碳。这四种人类生物素依赖的羧基酶都是大的多酶复合体，在奇链脂肪酸的氧化、支链氨基酸的分解代谢、脂肪酸的合成和糖异生等代谢途径中发挥着核心作用。三个人类生物素依赖的羧基酶基因的突变与酶缺乏以及由此导致的代谢和发育障碍丙酸血症、3-甲基巴豆酸甘氨酸尿症和乳酸酸血症有关。通过研究这些多酶复合体的组织、对酶功能重要的活性部位特征和残基，以及在缺乏患者中发现的错义突变可能造成的结构和功能后果，这些酶的结构-功能研究对于理解组装和催化的机制非常有价值。由于其相对容易分离和可用于体外研究，丙酸细菌的转羧酶多酶复合体长期以来一直被用作人类生物素依赖的羧基酶的模型系统。本应用重点研究细菌转羧酶和两种人类酶，丙酰辅酶A羧基酶和甲基巴豆酰辅酶A羧基酶的结构和功能。广泛的目标是对这些酶的结构和功能进行研究，以便更好地了解它们作为多酶复合体的组装及其催化活性的机制。具体目的是：1.采用X射线结晶学和诱变相结合的方法，分别研究生物素转羧酶12S和5S亚基的羧化和脱羧化机理。2.采用生物物理和生物化学相结合的方法，探讨转羧酶5S亚基催化的羧基转移酶反应中活性部位赖氨酸氨甲基化的相关性。3.寻求多亚基形式的转羧酶和人用酶丙酰-辅酶A和甲基巴豆酰-辅酶A的高分辨结构。这些结构将提供前所未有的亚单位-亚单位界面和人类酶的细节的视图，但也将作为更准确的模板，用于计算模拟人类酸血症突变可能产生的分子后果。生物素依赖的酶在人类新陈代谢中非常重要。在代谢和发育障碍患者中发现了改变其基因的突变。研究这些酶的结构-功能关系将有助于理解它们是如何组装和功能的，以及突变可能如何导致疾病。

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.