Title: Structural and functional studies on the regulation of pyruvate carboxylase by the bacterial second messenger cyclic-di-AMP
Description: The primary focus of this dissertation is the metabolic enzyme pyruvate carboxylase (PC). The structure and function of this fascinating enzyme has been studies and characterized by many laboratories over many decades. This extensive background is reviewed in Chapter 1, with an overview of the biotin-dependent carboxylase family and a particular focus on PC. In this dissertation, we primarily use X-ray crystallography to study PC at a structural level. This dissertation is divided into two overarching sections, with the first section (Chapters 2-5) focusing on the bacterial second messenger cyclic-di-AMP (c-di-AMP). This project was initiated by our collaborators in the laboratory of Josh Woodward at the University of Washington, who performed the first screen to identify c-di-AMP binding proteins in the bacterium Listeria monocytogenes. In Chapters 2 and 3, the regulation of PC by c-di-AMP in L. monocytogenes as well as the bacterium Lactococcus lactis is discussed. Crystal structures of the PC from each of these species in complex with cyclic-di-AMP reveal the binding site and give insights into the molecular mechanisms of this regulation.

In Chapters 4 and 5, structural studies of other c-di-AMP binding proteins identified in the screen are discussed. The second section (Chapters 6) focuses on a second class of PC enzymes called the two-subunit PCs, which are found in a subset of Gram-negative bacteria. In Chapter 6, the first crystal structure of a two-subunit PC from the bacterium Methylobacillus flagellatus is determined. In collaboration with the Lars Dietrich laboratory at Columbia University, we investigate the physiological function of the two-subunit PC in the pathogen Pseudomonas aeruginosa. A theme which emerges from these studies is that PC is an incredibly diverse enzyme which has been adapted for the peculiar physiological needs of each organism it inhabits. Because PC is found throughout nature in every kingdom of life, further studies of its unique properties and role in each organism are sure to provide more surprising insights in the years to come.