Enzymes that hydrolyze beta-lactams and cephalosporin antibiotics are called beta-lactamases, and their production by bacteria is the most common way that bacteria become antibiotic-resistant. The Zn(II)- containing beta-lactamases constitute an ever-growing and troubling class of beta-lactamases which contain 2 moles of Zn(II) per mole of enzyme, hydrolyze all known penicillin and cephalosporin antibiotics, are not inhibited by clavulanic acid, and have no known inhibitor of activity. The long-term objective of the proposed research is the rational design and preparation of irreversible inhibitors of the Zn(II)-containing beta-lactamases. Given the apparent structural and mechanistic differences among the metallo-beta-lactamases, this objective can only be realized after detailed characterization of a member from each distinct subclass of metallo-beta-lactamases. Similarities between the enzymes can then be identified and exploited for the rational design of an inhibitor. This proposal involves the study of the structurally-distinct, biomedically-important beta- lactamase (L1) from X maltophilia.
The specific aims of this proposed research are: (1) detailed structural characterization of the metal binding sites of metal-substituted forms of the X. maltophilia beta- lactamase using spectroscopic, mutagenesis, and binding studies, (2) elucidation of the mode of action of the enzyme with several penicillin and cephalosporin antibiotics using steady-state and pre-steady state kinetics studies, (3) identification of the overall reaction mechanism used by Ll and of any stable reaction intermediates using mathematical simulations of kinetics rate profiles and results from biochemical studies, (4) integration of structural and mechanistic results to identify key aspects of the enzyme that can be targeted for the development of novel reaction- or structure-based inhibitors, and (5) examination of the possible inhibition properties of compounds, when once hydrolyzed produce reactive, exocyclic methylene groups. It is hoped that this novel integration of kinetics, biochemical, simulations, and spectroscopic studies can be used as a general strategy for the preparation of new antimicrobial agents and a better way to combat the ever-increasing prevalence of antibiotic resistance in bacteria.
