Biochemical reconstitution and characterization of peptidoglycan synthases and glycosidases

Abstract

The peptidoglycan cell wall is a unique macromolecular structure in bacteria that is essential for their survival and morphogenesis. The peptidoglycan synthesis pathway is an excellent target for antibiotics, and decades of research have focused on characterizing this pathway. For many years, it was thought that only penicillin-binding proteins (PBPs) and related enzymes were able to synthesize peptidoglycan. Here, I show that FtsW, which is an essential cell division protein that belongs to the SEDS (shape, elongation, division and sporulation) family of proteins, is a peptidoglycan polymerase. Biochemical reconstitution revealed that this activity is dependent on complex formation with its cognate class B PBP (bPBP), and cellular studies demonstrated that a functional FtsW is required for viability. Our findings establish FtsW as a key peptidoglycan polymerase involved in septal cell wall assembly. Next, I report a high-throughput assay that allows measurement of peptidoglycan polymerase activity. Peptidoglycan polymerases are appealing targets for antibiotic development, but inhibitor discovery has historically been hampered by a lack of adequate assays to assess polymerization. Using the newly developed assay, I conducted a small-scale screening campaign of a Staphylococcus aureus lethal compound library and identified small molecules that inhibit S. aureus FtsW activity in vitro. In the final section of this dissertation, I present the biochemical characterization of MpgA and MpgB, which are membrane-bound proteins that are important for Streptococcus pneumoniae cell wall assembly. These enzymes were predicted to be peptidoglycan glycosidases that perform different chemical reactions, but I show that both enzymes are peptidoglycan muramidases. MpgA and MpgB cut the peptidoglycan backbone at different sites, and I identify the mechanistic basis for cleavage site selection as well as an amino acid switch that alters the cleavage chemistry. I propose that these muramidases function as peptidoglycan release factors that liberate newly synthesized peptidoglycan from the membrane. Collectively, my work demonstrates the utility of biochemical reconstitution in understanding how bacteria build their cell wall and lays the groundwork for discovering new antibiotics that target this process.