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 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 hydrolysis of ceftotaxime by CTX-M enzymes as well as 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. 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. However, the extent to which the active site functions as a highly cooperative, interconnected unit versus the extent to which it is modular is unknown. 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. It is not known, however, how KPC-2 is able to hydrolyze carbapenems while other class A ?-lactamases, although similar in structure to KPC-2, cannot. An unbiased genetic approach will be used to identify mutants specifically defective in carbapenem hydrolysis. This approach has the advantage of surveying for changes anywhere in the enzyme that decrease carbapenem hydrolysis. Detailed biochemical and structural characterization of the mutant enzymes will address the mechanism behind the unique catalytic properties of class A carbapenemases.
?-lactamases are enzymes that hydrolyze ?-lactam antibiotics to provide bacterial drug resistance and the emergence of ?-lactamases that are capable of hydrolyzing virtually all ?-lactam antibiotics is a significant threat to the efficacy of antibiotic therapy. The proposed experiments utilize genetic analysis, enzyme kinetics, X-ray crystallography and high throughput sequencing of combinatorial amino acid substitution libraries to study the structure and function of two clinically important ?-lactamases. The results will provide detailed knowledge of how amino acid residue positions contribute to ?-lactam hydrolysis, which will facilitate understanding the molecular basis of the evolution of antibiotic resistance.
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