Bacterial resistance to antimicrobial agents has increased in recent years, posing a significant threat to antibiotic therapy. ?-lactam antibiotics are the most often prescribed antibacterial agents. The most common mechanism of resistance is ?-lactamase-catalyzed hydrolysis, which renders the antibiotics ineffective. Because of the diverse range of substrate specificities of these enzymes, virtually all ?-lactam antibiotics are susceptible to hydrolysis. The design of new antibiotics that escape hydrolysis by the growing collection of ?-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of these enzymes. The objective of this application is to understand how amino acid sequence determines the structure, activity and evolution of class A ?-lactamases. The CTX-M ?-lactamases comprise a diverse family of extended-spectrum ?-lactamases (ESBLs) that efficiently hydrolyze the oxyimino-cephalosporin cefotaxime but not ceftazidime. Certain amino acid substitutions in resistant clinical isolates, however, have been associated with increased ceftazidime hydrolysis. The goal of Aim 1 is to understand the structural and biochemical basis for the altered catalytic activity observed in CTX-M variants. In addition, studies from the current funding period indicate that cooperative interactions between amino acid residues in and near the active site play an important role in determining CTX-M substrate specificity and driving molecular evolution. How active site residues cooperate to catalyze reactions is an important question with regard to how enzymes function. It is well-established that certain active site residues work together, cooperatively, to achieve catalysis. The general extent of cooperativity among active site residues, however, has not been evaluated systematically. The extent of cooperativity between substitutions will be addressed systematically in Aim 2 using random mutagenesis of active site residues of CTX-M-14 in combination with deep sequencing of mutants selected for the ability to hydrolyze various ?-lactam antibiotics. Finally, KPC-2 ?-lactamase is able to hydrolyze nearly all ?-lactam antibiotics including carbapenems but not ceftazidime. Nevertheless, natural variants of KPC-2 have been found that exhibit increased ceftazidime hydrolysis without losing the capacity to hydrolyze carbapenems. It is not known, however, how the substitutions in these variants alter the structure and catalytic properties of KPC-2 to bring about the change in substrate specificity. This question will be addressed in Aim 3 using a combination of enzyme kinetics analysis and X-ray crystallography.