The bacterial acquisition of beta-lactamases is the primary mechanism for their resistance to beta-lactam antibacterials (e.g., penicillins and cephalosporins). A multidisciplinary approach has been outlined for the study of the TEM-1 beta-lactamase, the prototypic member of the class A beta-lactamases. The proposed research benefits from the applicant's expertise in protein chemistry, organic synthesis, microbiology, and molecular biology. Crystallographic data and molecular modeling of penicillin and cephalosporin binding into the active site of the TEM-1 enzyme suggests the involvement of a hydrogen bond by the side chain of Asn-132. The role of Asn-132 residue in catalytic turnover will be probed by site-directed mutagenesis and specially designed substrates. The mechanistic function of Lys-73 which is highly conserved among class A beta-lactamases is unclear. Straight-forward analysis of mutants at this position would not provide a conclusive answer, so a novel NMR experiment is proposed to elucidate the function of this residue. Non-classical beta-lactam structures such as carbapenems are resistant to hydrolytic deactivation by beta-lactamases but the mechanistic basis is not understood. Molecular modeling has provided mechanistic insight into the process of turnover of carbapenems which may help. Based on these findings, new beta-lactamases molecules are proposed which are expected to be poor substrates for these enzymes, but are predicted to retain antibacterial property. Three new classes of mechanism-based inactivators have been proposed for beta-lactamases. These molecules will be synthesized and studied in detail as potential inactivators for both TEM-1 Q908R (class C) beta-lactamses. A novel cephalosporin has been designed which may be useful in delineating the structural basis for evolutionary kinship of penicillin-binding proteins (PBPs) and beta-lactamases.
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