Characterization of peptidoglycan glycosyltransferases: Essential enzymes for bacterial cell wall biosynthesis

Peptidoglycan is a macromolecule that surrounds bacteria and is essential for their survival. Peptidoglycan glycosyltransferases (PGTs) are responsible for the extracellular assembly of the glycan strands that make up the sugar backbone of the peptidoglycan layers. Compromising the integrity of the peptidoglycan layers is a good means to effectively kill bacteria. Given their essential role in the extracellular steps of the pathway, PGTs are particularly good targets for the development of new antibiotics to treat resistant bacterial infections. Much progress has been made in understanding earlier enzymes in the biosynthetic pathway; however, for several reasons progress in studying PGTS has been relatively slow. Studies of the PGTs were impeded by limited access to substrates for the development of in vitro assays, challenges in obtaining model PGTs and difficulties in characterizing the products of the in vitro reactions.; Synthetic routes to PGT substrates have been achieved and this thesis describes the expression and characterization of three PGTs from three different bacteria: E. coli, Staphylococcus aureus, and Aquifex aeolicus. The biochemical properties and kinetic parameters of the three PGTs are discussed. These enzymes show some differences in their activity and behavior, which may reflect differences in their roles in bacteria.; This thesis also describes the development of a SDS-PAGE method to analyze product distributions from PGT reactions. Using this method it was shown that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for PGTs, but that the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. The SDS-PAGE method can also be applied to provide information on the roles of invariant PGT residues in catalysis. A comprehensive mutational analysis of the A. aeolicus PGT domain shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described herein provide insights into the mechanism of PGTs and allows for the investigation of factors that can affect polymerase processivity.