Structural and Functional Studies of Biotin-Dependent Carboxylases

A persisting question in biology concerns the exceptional diversity of metabolic enzymes and how they respond to their ligands and dynamic environments with remarkable precision. In humans, the family of biotin-dependent carboxylases holds important roles in intermediary metabolism. Recent years have witnessed significant progress toward understanding these enzymes' roles in homeostatic regulation. However, due to a lack of structural information, their catalytic mechanisms, as well as the macromolecular consequences of their genetic mutations, are still not well understood. This dissertation describes the characterization of two biotin-dependent carboxylases that catalyze essential metabolic transformations in humans and bacteria, using X-ray crystallography to elucidate their structures and biochemical assays to verify their activities. We engineer a novel chimeric variant of propionyl-CoA carboxylase (PCC) and produce the first crystal structure of its 750-kDa α6β6 holoenzyme. This structure reveals the architecture of PCC's twelve catalytic domains and allows the mapping of its disease-associated gene mutations to predict their effects on enzyme stability and catalysis. We also identify and describe a new domain that is integral to maintaining inter-subunit contacts within PCC. Following this, we extend our studies to methylcrotonyl-CoA carboxylase (MCC), another 750-kDa α6β6 holoenzyme that differs from PCC primarily in its substrate preference. The crystal structure of MCC assumes a markedly different configuration from PCC despite the high sequence identity between the two. Theorizing that these enzymes may represent unique lineages in the evolution of the biotin-dependent carboxylases, we apply similar approaches to the study of a third biotin-dependent carboxylase. Our efforts have produced the first two holoenzyme structures of CoA-recognizing biotin-dependent carboxylases, and provide valuable insight for understanding the functions of these vital enzymes.