Crystallographic studies of bacterial proteins involved in beta-lactam resistance

The β-lactams are an important class of antibiotics that target penicillin binding proteins (PBPs), bacterial enzymes that catalyze the final step of cell wall synthesis. The bacterial cell wall maintains cell shape and provides protection against osmotic lysis. PBPs catalyze the formation of peptide crosslinks in peptidoglycan, which are essential to the structural integrity of the cell wall. Due to the uniqueness of the peptidoglycan of bacterial cells (with no biochemical equivalent in humans), β-lactams have been highly successful in treating bacterial infections. Unfortunately bacteria have developed multiple resistance mechanisms that have substantially reduced the effectiveness of this class of antibiotics. Three main mechanisms of β-lactam resistance have been found in resistant bacteria: production of β-lactamase enzymes that hydrolyze and inactivate β-lactams, over-expression of multi-drug efflux pumps in Gram-negative bacteria that actively pump out β-lactams and other antibiotics, and the expression of resistant PBPs with unusually low affinities for β-lactams. The overall goal of this thesis is to use the technique of X-ray crystallography for the structural characterization of specific proteins involved in each of these three main mechanisms.; PSE-4 is a class A β-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild type PSE-4 carbenicillinase has been determined to 1.95 Å resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. Most carbenicillinases are unique among class A β-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50 fold reduction in k_cat/K _m and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 Å resolution with the wild type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A β-lactamase structures. The molecular modelling studies presented here suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin α-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild type PSE-4 may be involved in accommodating this shift in the bound substrate position.; MexR is a member of the MarR family of bacterial transcriptional regulators and is the repressor for the MexAB-OprM operon, which encodes a tripartite multidrug efflux system in Pseudomonas aeruginosa. Mutations in MexR result in increased resistance to multiple antibiotics due to over-expression of this efflux system. (Abstract shortened by UMI.)...