The murein sacculus is a mesh of cross-linked peptidoglycan strands that confers rigidity to the bacterial cell wall. Beta-lactam antibiotics, which target the essential transpeptidases (penicillin-binding proteins or PBPs) that cross-link the peptidoglycan strands, are important compounds in the treatment of bacterial diseases. Unfortunately, the emergence of multiple mechanisms of antibiotic resistance threatens to make these and other antibiotics obsolete in the treatment of bacterial infections. Along with other pathogenic bacteria, antibiotic resistance in Neisseria gonorrhoeae is a growing problem. Penicillin and tetracycline, once the antibiotics of choice for treatment of gonococcal infections, are no longer be used due to the emergence of resistant strains. Moreover, increasing numbers of strains are now resistant to the fluoroquinolones, one of the two antibiotics current recommended in the treatment of gonorrhea. Clearly there is an urgent need to develop new antimicrobials directed both against well-known molecular targets, such as PBPs, but also against novel targets. In this proposal we describe structural and biochemical studies of three enzymes involved in peptidoglycan metabolism: a D-D-carboxypeptidase from E. coli (PBP 5) that serves as a model system for elucidating PBP function, an essential transpeptidase (PBP 2) from N. gonorrhoeae that is the lethal target of current beta-lactam antibiotics, and a lytic transglycosylase, MltA, also from N. gonorrhoeae, that serves as the lynchpin of the cell wall synthesizing complex. Each of these proteins has been selected to address one or more of the following aims: (a) to understand the biology of peptidoglycan synthesis, (b) to explore their interactions with antibiotics, (c) to elucidate the molecular basis for antibiotic resistance and (d) to examine their potential as targets for drug development. Studies on PBP 5 will elucidate the mechanism by which this enzyme hydrolyzes substrate and will provide a better understanding of PBP-antibiotic interactions in general. The molecular basis for antibiotic resistance in PBP 2 will be investigated by structural studies of the native enzyme and of a mutant isolated from a penicillin-resistant strain. The role of MltA as part of a multienzyme complex mediating peptidoglycan synthesis as well as its suitability as a novel target for antimicrobials will be examined by solving its crystal structure. These studies will provide a framework for future studies aimed at structure-based drug design and will provide substantial insight into the mechanisms of peptidoglycan synthesis. ? ?
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