Faculty Scholarly Dissemination Grants

Title

Factors Determining Carbapenemase Activity in the OXA Family of ²-Lactamases

Department

Cell and Molecular Biology

College

College of Liberal Arts and Sciences

Disciplines

Life Sciences

Abstract

Bacterial resistance to antibiotic therapies, especially to ²-lactams is a growing health-care problem. Resistance mediated by class D ²-lactamases has been both the least studied, and most rapidly expanding in the past decade. Of particular clinical concern is the emergence of class D enzymes with the ability to hydrolyze the newest family of ²-lactams: the carbapenems. Class D ²-lactamases are extremely diverse in terms of sequence and hydrolytic profiles, and it remains unclear what factors determine multispecificity in general, and carbapenemase activity in particular. Recent studies have revealed the importance of the ²5-²6 loop in acquiring carbapenemase activity1. Here we present a combined experimental and computational study of the effects that several point mutations in the OXA-24s ²5-²6 loop have on the enzyme catalytic profile. Site-directed mutagenesis and kinetic assays indeed show significant changes in the catalytic profiles of the mutant enzymes. We have employed several computational techniques, namely sequence and motif analysis, Molecular Dynamics simulations and covariance analysis based on the Anisotropic Network Model in order to determine the impact of M223A, G224D and P227S mutations on the dynamics of the OXA-24 active site. We show here that the mutations affect the dynamics of the catalytic site, specifically the carboxylated lysine residue and its hydrogen bonding network within the binding pocket. Multiple sequence alignment and motif analysis show distinct patterns of the ²5-²6 loop sequence variation in different subgroups of OXA carbapenemases. These data will help correlate the sequence traits of OXA carbapenemases to their mechanism of substrate selection and hydrolysis.

Conference Name

27th Annual Symposium of the Protein Society

Conference Location

Boston

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