Date Approved

4-2015

Graduate Degree Type

Thesis

Degree Name

Cell and Molecular Biology (M.S.)

Degree Program

Cell and Molecular Biology

First Advisor

Agnieszka Szarecka

Abstract

Bacterial resistance to antibiotic therapies, especially to β-lactams, is an increasing problem. β-lactamases are the main source of clinical resistance to these antibiotics, and the class D β-lactamases are one of the most rapidly expanding classes of these enzymes. The emergence of class D enzymes with the ability to hydrolyze the newest family of β-lactams - the carbapenems - is a serious concern for the healthcare system as carbapenems are last resort antibiotics: ideal for severe infections after other therapies have failed. Class D β-lactamases are very diverse in terms of sequence and substrate profile, and it remains unclear what factors affect the enzymes’ ability to hydrolyze certain classes of antibiotics (e.g. carbapenems or cephalosporins). The β5-β6 loop has been previously shown to affect the substrate profile of OXA enzymes, for carbapenems in particular. This work examines the effects of selected mutations in the β5-β6 loop on the proteins’ dynamics via Molecular Dynamics simulations. OXA-24 (a carbapenemase) and three mutants (M223A, G224D, P227S) were simulated for 40 nsec, and the trajectories revealed that all three mutations alter the dynamics of the enzyme. Our data show that the mutations affect the flexibility of several crucial segments of the enzyme structure, the overall compactness of the protein, as well as the size of the active site. Our results suggest that the β5-β6 loop can affect the substrate profile of OXA-24 by modulating the enzyme’s dynamics in a way that is consistent with substrate profile expansion, in particular with the ability to bind 3rd generation cephalosporins.

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