) Penicillin-binding proteins (PBPs) are a group of enzymes involved in a number of functions in the assembly and regulation of bacterial cell wall. These enzymes are the targets of beta-lactam antibiotics for inhibition of bacterial growth. A multidisciplinary approach has been outlined for the study of PBPs, which builds on the mechanistic findings from this laboratory presented as Preliminary Results.
Pour Specific Aims are outlined.
Specific Aim 1 details the plans for cloning, expression and large-scale production of two PBPs, one from Escherichia coli (a Gram-negative bacterium) and another from Staphylococcus aureus (a Gram-positive bacterium). These proteins will be used in the biochemical studies and also will be provided to Professor Judy Kelly of the University of Connecticut for crystallization.
Specific Aim 2 describes our design and proposed syntheses for two cephalosporins that are incorporated with structural components of the cell wall (peptidogylcan). These cephalosporins, in conjunction with one that is already synthesized, are proposed as mechanistic probes for the transpeptidase reaction carried out by certain PBP in the last step of cell wall biosynthesis (cross-linking of cell wall). Biochemical and structural experiments are detailed for the use of these cephalosporins as probes of mechanisms for PBPs. An assay for the cell wall cross-linking reaction of the transpeptidases (a PBP) is described in Specific Aim 3. The enzymic reaction is biochemically dissected into the acylation and deacylation steps, for each of which a quantitative assay method is described. These methodologies will allow investigations of the mechanistic details of these PBPs. Furthermore, a series of four peptidoglycan derivatives have been proposed to investigate the requirements for a minimal substrate for the transpeptidation reaction of the PBPs.
Specific Aim 4 details the search for novel non-f3-lactam inhibitors for PBPs. These molecules will be synthesized and their potential PBP inhibitory and antibacterial activities will be investigated in both in vivo and in vitro experiments.
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