STRUCTURAL STUDIES OF BACTERIAL METALLO-BETA-LACTAMASES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial resistance to the beta-lactam family of antibiotics (penicillins and cephalosporins) has progressed at a tremendous rate since it first appeared clinically several years ago. Counteraction of the drugs is accomplished by the expression of extracellular enzymes which hydrolyze the N-C(=O) lactam bond. Approximately 20 of the known beta-lactamases require one or two equivalents of Zn(II) for activity. Of these, the majority reach maximal activity with two equivalents of Zn(II). However, three metallo-beta-lactamases have been shown to be fully active with one Zn(II) bound, and these are inhibited by binding of a second Zn(II). Several metallo-beta-lactamases have been isolated from human pathogens, and to date there is no clinically viable inhibitor available. Our purpose is to use XAS to complement magnetic resonance experiments, in combination with rapid-freeze-quench techniques, to characterize stable resting states and catalytic intermediates relevant to metallo-beta-lactamase catalyzed hydrolysis. The short-term goal is to determine the structural features that govern metallo-beta-lactamase activity. Our long-term focus is a detailed understanding of the factors controlling reactivity that are determined by the second metal ion.