Peptidoglycan Fragments to Understand Transpeptidases – PFUT

Penicillin-binding proteins (PBPs) are the targets of beta-lactams, the most widely used antibiotics such as penicillins, cephalosporins or carbapenems. PBPs are transpeptidases that catalyze the cross-linking of the peptidoglycan (PG), the main component of the bacterial cell wall. Pathogens such as Staphylococcus aureus, enterococci, Neisseria ssp. and Streptococcus pneumoniae become resistant to penicillins by expressing PBPs with a low reactivity for beta-lactams. Since these drugs are structural mimics of the natural substrate, how PBPs from resistant strains have lost their reactivity with beta-lactams while retaining their enzymatic function? The solution to this conundrum will explain why some last generation beta-lactams are active against strains resistant to older drugs, and help design further improvements.
Although the reaction between PBPs and beta-lactams is well understood kinetically and structurally, the enzymatic transpeptidation catalyzed by PBPs is poorly studied due to the difficulty to obtain the substrates. The transpeptidation occurs between two stem-peptides attached to glycan chains. However, the peptides on their own or attached to disaccharides are not substrates of PBPs in vitro. Progress in the enzymology of PBPs have recently uncovered some requirements of the substrates of the transpeptidation reaction, using glycan chains polymerized in vitro. However, structural information about the interaction between PBPs and their substrates are still lacking despite half a century of trying, because the polymerization of glycan chains that can be achieved in vitro yields mixtures heterogenous in size, precluding crystallographic studies and detailed enzymology.
We have demonstrated a chemo-enzymatic approach that allows to synthesize PG fragments of defined size using a glycosynthase engineered by mutation of hen egg-white lysozyme. The precursors are obtained after accumulation in specially engineered strains of Escherichia coli that highjack the PG recycling pathway.
We propose to use these uniquely available PG fragments to define structurally for the first time the interaction between PBPs and their substrates, by combining x-ray crystallography and NMR with enzymology and binding studies. Our target enzymes will be PBP2x, PBP2b and PBP1a from Streptococcus pneumoniae. These three PBPs are involved in the resistance to beta-lactams of this important human pathogen and we will investigate variants from a susceptible and a highly resistant strain. The interaction of these PBPs with beta-lactams has been well characterized, structurally and kinetically, but information on the interaction with their physiological substrates is still lacking.
The use of novel well-defined substrate analogues will provide for the first time a detailed understanding of the important drug targets that are the PBPs.