This application is concerned with the crystallographic aspects of a collaborative effort to study the origin of bacterial resistance to beta- lactam antibiotics related to beta-lactamase. The other parts of the collaboration include the molecular biology of beta-lactamase, in particular site directed mutagenesis, the biochemistry aspects, and the molecular modeling of the system. The study is relevant to the recovery of the effectiveness of penicillin and cephalosporin therapy, which has been rapidly diminishing due to the ability of pathogenic bacteria to produce beta-lactamases. These enzymes hydrolyze the beta-lactam ring prior to the delivery of the drug to its target - the bacterial cell wall - where the penicillin sensitive enzymes responsible for the cell wall synthesis and repair are located. The understanding of the catalytic process and the substrate specificity at the atomic level is required for the development of new effective antibiotics. X-ray crystallography provides such accurate information. The high resolution crystal structure of beta-lactamase from Staphylococcus aureus PC1 enables us to carry out binding studies to various substrates and inhibitors. Cryocrystallography, aiming at trapping reaction intermediates at liquid nitrogen temperature, will be employed, as well as conventional room temperature measurements. Site directed mutants have been designed to test the proposed catalytic mechanism, alter substrate specificity, and probe the folding properties of the enzyme. These will be prepared for biochemical and crystallographic analysis. To address the problem of the recently acquired resistance to the potent """"""""third generation"""""""" cephalosporins, the crystal structures of the E. coli SHV1 and SHV2 beta-lactamases will be determined, the latter being responsible for the new resistance. The various aspects of the project will provide information pertaining not just to the specific clinical problem caused by beta-lactamases, but will also contribute to our fundamental understanding of protein function and stability.
Showing the most recent 10 out of 13 publications
