EXCEEDTHE SPACE PROVIDED. P-lactam antibiotics such as the penicillins and cephalosporins are among the most often prescribed antimicrobial agents. These antibiotics act by inhibiting transpeptidase enzymes (also called penicillin binding proteins or PBP's) that are essential for the synthesis of the peptidoglycan layer of the bacterial cell wall. Inhibition of peptidoglycan synthesis results in death of growing bacteria and accounts for the antimicrobial effect of P-lactam antibiotics. In response, bacteria have evolved defense mechanisms to resist the lethal effects of these drugs. Due to widespread p-lactam antimicrobial use, bacterial resistance has increased and now represents a serious threat to human health. The most common mechanism of resistance to P-lactam antibiotics is the production of p-lactamases, which cleave the antibiotic, rendering it ineffective, p-lactamases are grouped into four classes (A,B>C, and D) based on primary amino acid sequence homology. The class B enzymes require zinc for activity and catalyze the hydrolysis of virtually all P-lactam antibiotics. The goal of this study is to understand how the amino acid sequence determines the structure and activity of the class B metallo-p-lactamases IMP-1 and Bla2. In the previous funding period we used in vitro mutagenesis to randomize codons encoding amino acid residues in the active site region of the IMP-1 enzyme. These random libraries were used to identify residues that alter the substrate specificity of the enzyme. A detailed study of the residue positions that control substrate specificity will be undertaken to understand the molecular basis of recognition and catalysis of p-lactam antibiotics. This study will also include characterization of inhibitors of the IMP-1 and Bla2 p-lactamases with respect to inhibition constants and mode of inhibition. A further objective is to continue our study of the determinants of binding specificity of the p-lactamase inhibitory protein, BLIP, and to use this information to engineer BLIP molecules that bind and inhibit P-lactamases and penicillin binding proteins (PBPs) from Gram-positive bacteria. The information gained from these studies will be useful for rational antibiotic and inhibitor design that takes into account the evolutionary capacity of the target enzyme.
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