The role of species-specific modifications in peptidoglycan biosynthesis

Penicillin binding proteins (PBPs) are responsible for the final extracellular steps transglycosylation and transpeptidation) in the biosynthesis of peptidoglycan, the essential cell-wall carbohydrate polymer, from its Lipid II precursor. They are excellent targets for antibiotics due to their essentiality for cell viability in most bacteria. Genus- and species-specific variation in the chemical structure of Lipid II can have significant consequences for the formation and metabolism of the peptidoglycan sacculus by the PBPs. Characterisation of these enzymes from Grampositive bacteria, including the substrate-enzyme interactions involved, is essential in both understanding the mechanisms of peptidoglycan biosynthesis and contributing to the development of new antimicrobials. \n \nThe work presented in this thesis focuses primarily on the substrate specificity of the Streptococcus pneumoniae bifunctional PBPs; PBP1a and PBP2a; both recombinantly expressed and purified to a high level. The preference, by both enzymes, for amidated Lipid II as a transglycosylase substrate was identified by two complementary assay systems. A novel spectrophotometric assay was used to observe transpeptidation by S. pneumoniae PBP1a in a continuous manner; the first time this has been achieved for a Gram-positive PBP, and with potentially farreaching implications in the future of antibiotic discovery. Attempts were made to synthesise dipeptide branched Lipid II, implicated in β-lactam resistance, as substrates for the bifunctional PBPs. The role of amidation in Staphylococcus aureus peptidoglycan biosynthesis was also investigated, and variation in the requirement for this modification between the monofunctional transglycosylase MGT and bifunctional PBP2 identified. Two novel monosaccharide compounds were identified as inhibitors of transglycosylation. \n \nThis thesis provides an important basis for understanding the peptidoglycan biosynthesis mechanisms of two globally important pathogens. This insight, and future work leading from it, could contribute to the development of new antibiotics, helping to reduce the global threat of antimicrobial resistance.