Electrostatics of Antibiotic Resistance-Measuring the Evolution of Electric Fields in beta-Lactamases Using the Vibrational Stark Effect

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Antibiotics saved countless lives and are indispensable agents in our health system. At the same time their extensive use and misuse led to the appearance of severe antibacterial resistances that are responsible for 15 000 death p.a. only in Germany and are thus regarded as a major public health thread by the German Federal Government. Beta-Lactam antibiotics are the most frequently used antibacterial drugs and aim at the inhibition of the cell wall-synthesis. However, the continual development of novel beta-lactams caused the emergence and mutational evolution of hundreds of interceptor proteins called beta-lactamases that confer resistance against these drugs. These proteins deactivate beta-lactam antibiotics via a hydrogen-bond (H-bond) mediated chemical mechanism: the formation of a beta-lactam acyl-enzyme ester and its subsequent hydrolysis. Only recently, it was shown that H-bonds exert immense electric fields and can provide a major driving force accelerating enzymatic reaction rates by 5 orders of magnitude. A similar situation is expected in beta-lactamases where very strong H-bonds, e.g. between the beta-lactam and oxyanion hole in the active sites, may uniquely power and thus regulate resistance to antibiotics. Intriguingly, beta-lactams have an intrinsic so-called Stark reporter group i.e. the beta-lactam carbonyl group that allows determining electric fields using the vibrational Stark effect. Using an interdisciplinary approach combining biology, vibrational spectroscopy, and theory, the proposed project aims at tracking the evolution of the electric fields within the active sites of beta-lactamases along the line of clinically documented mutants responsible for the emergence of antibiotic resistances. Specifically, this work will focus on the evolutionary pathway TEM-1 - TEM-12 - TEM-10 - TEM-5 (CAZ-1) leading to extended-spectrum beta-lactamases (ESBL) that already hydrolyse antibiotics such as 3rd generation cephalosporins. The knowledge obtained from this project is of highest biomedical relevance and can help tackling the global thread of antimicrobial resistance.