Research Project
8287854
DESCRIPTION (provided by applicant): Carbapenem antibiotics such as doripenem play a critical role in the treatment of life-threatening bacterial infections. The emergence of bacterial ?-lactamase enzymes that can break-down and incapacitate these important drugs threatens their therapeutic value. We plan to study how one such carbapenemase enzyme, OXA-24, is able to bind and hydrolyze carbapenem substrates. Current results from our lab and others suggests that after a carbapenem (eg. doripenem) binds to a carbapenemase enzyme, the drug undergoes one or more conformational changes that determine whether it will be fully hydrolyzed or stay in the active site as an inhibitor. In our proposed studies we will use mutagenesis, minimum inhibitory concentration analysis, kinetic assays and X-ray crystallography to understand how the conformation of the drug changes as it binds and undergoes chemical bond cleavage. We will investigate which active site amino acid residues in OXA-24 are responsible for conformational changes such as hydroxyethyl rotation and post-acylation tautomerization, and how substitutions at those positions affect overall rates of hydrolysis. OXA-24 is an excellent model system for studying class D carbapenemase enzymes; it is easy to purify and crystallize for structural studies, and the fact that it is a monomer simplifies any kinetic analysis. OXA-24 also contains 8-9 active site residues that are highly conserved among class D ? -lactamases, so the results we obtain will be broadly applicable. Ultimately, by discovering the details of carbapenem hydrolysis on these enzymes, we hope to provide direction for the design of more effective antibiotics and ? -lactamase inhibitors. 1 PUBLIC HEALTH RELEVANCE: Bacterial resistance to ? -lactam antibiotics continues to grow at an alarming rate, rapidly threatening the efficacy of old and new treatments alike. Class D ? -lactamases in particular are emerging in problematic Gram-negative species such as Acinetobacter baumannii, and they pose a serious threat to the use of the four clinically-approved carbapenems: imipenem, doripenem, meropenem and ertapenem. OXA-24 is one of the most prevalent class D carbapenemases and serves as an excellent model enzyme. A detailed understanding of how class D ? -lactamases bind and hydrolyze carbapenems will aid in the design of more effective antibiotics and ? -lactamase inhibitors. 1
