Infections caused by gram-negative bacteria are an increasing health problem, particularly in hospitalized patients. Plasmids in these organisms determine a variety of beta-lactamases, enzymes that are responsible for resistance to penicillin, cephalosporin, and related beta-lactam antibiotics. More than 30 plasmid-mediated beta-lactamases have been distinguished, and in some cases new enzymes with expanded specificity seem to be evolving from established types. As the major biochemical mechanism for resistance to beta-lactam antibiotics it is important to be able to follow the distribution of these enzymes in gram-negative pathogens and to understand how these enzymes are related genetically, what structural features are responsible for their ability to hydrolyze particular beta-lactams, and what is their further potential for evolution in response to the changing selection pressure imposed by the introduction of new beta-lactam drugs.
The aims of this proposal are: 1. To establish the nucleotide sequence for PSE-l and OXA-7 beta- lactamases, two enzymes with distinctive properties whose structural basis will be clarified by comparison with the sequence of FSE-2 which we have determined and with OXA enzymes sequenced by others. 2. To construct enzyme hybrids between PSE-2 and OXA-5 or OXA-2 such that properties of the chimeric beta-lactamases, will aid in localization of particular enzyme functions. 3. To establish the structural basis for extended spectrum beta- lactamase activity in mutants and in a plasmid-mediated enzyme isolated at our hospital that confers resistance to aztreonam and ceftazidime. 4. To perfect conditions for selecting beta-lactamase mutants resistant to inhibition by clavulanic acid, sulbactam, or YTR 830 and to characterize those we have already obtained for the particular amino acid changes involved in diminished inhibitor affinity. 5. To analyze the contribution of amino acids making up the walls of the enzyme active site to substrate specificity by systematic site-directed mutagenesis.
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