The susceptibility of pathogenic bacteria to penicillins and related compounds has been greatly reduced because of the production of beta-lactamases, a group of enzymes that hydrolyze the beta-lactam amide bond characteristic of these antibiotics. The rapid increase in multi-resistant infectious bacteria, together with the prevalence of patients whose immune system has been compromised underscores the urgency of recovering the effectiveness of antibiotics in general, and of beta-lactam therapy in particular. Future design of novel drugs will benefit from the understanding o the mechanism of two enzyme families: the beta- lactamases - the penicillin-degrading enzymes; and the peptidases involved in bacterial cell-wall synthesis and repair - the penicillin-binding proteins. The plasmid mediated class A beta-lactamases have emerged in recent years as the group of enzymes that evolve most rapidly when beta-lactam antibiotics are introduced. The proposed studies will investigate the structural basis for the activity an evolution of these enzymes, using the class A beta-lactamase from Staphylococcus aureus PC1 as a model system. Questions about the catalytic mechanism, substrate specificity, and stability of the enzyme will be addresse by engineering variant molecules and analyzing them by biochemical and X-ray crystallographic methods. The structures of acyl-enzyme complexes with representative substrates will be determined so that insight into the basis fo substrate specificity at the atomic level will be gained. The evolutionary lin between the class A and the class C beta-lactamases, and between these beta-lactamases and the cell-wall peptidases will be investigated by designing specific changes that will convert the protein from a class A enzyme into one of the other related enzyme families. The design is structurally driven, based on analysis of crystal structures of representative members of each family. A mutant beta-lactamase that has lost its ability to hydrolyze beta-lactams and instead is inhibited by these compounds will be further altered to introduce a new deacylation mechanism that resembles that of the class C beta-lactamases. This mutant and the native proteins will serve as the parent molecules for engineering a carboxypeptidase activity toward D-Ala-D-ala peptide, the natura substrate of the bacterial cell wall peptidases.
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