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. ? ? ? ?
