Structural and Biochemical Studies of Enzymes in Bacterial Glycobiology

Abstract

The speed that bacterial pathogens gain resistance to antibiotics is alarming. Designing new antibacterial agents is urgent, but it requires understanding their bacterial targets at the molecular level to achieve high specificity and potency. In this thesis, I discuss the structural and biochemical investigations of three potential protein targets for antibiotics. The first is a UDP-Glc/GlcNAc 4-epimerase, called Gne, from the human pathogen Campylobacter jejuni. This enzyme is the sole source of N-acetylgalactosamine (GalNAc) in C. jejuni, which is a common component in three major glycoconjugates decorating the cell surface and is critical for pathogenesis. The second target protein is an integral membrane protein, called MraY, which catalyzes the transfer of phospho-N-acetylmuramyl (MurNAc) pentapeptide to a lipid carrier, undecaprenyl phosphate (C55-P), producing Lipid I in the peptidoglycan biosynthesis pathway. In the following step, a peripheral protein called MurG catalyzes transferring N-acetylglucosamine (GlcNAc) to Lipid I and produces Lipid II, which provides the first building block of the peptidoglycan layer. Peptidoglycan is uniquely bacterial, with MraY and MurG both being essential for cell viability; therefore, they are attractive targets for the development of antibacterial agents and work toward their structures is presented. Finally, MraY from Escherichia coli is the target for the lysis protein E from phage ΦX174.Efforts toward elucidating the EcMraY-E complexstructure are demonstrated here. In total, this thesis provides important data toward a full mechanistic understanding of these important antibacterial targets.