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

抗生素拯救了无数人的生命，是我们卫生系统中不可或缺的媒介。与此同时，它们的广泛使用和误用导致了严重的抗菌素耐药性的出现，每年导致15000人死亡。仅在德国，因此被德国联邦政府视为一条主要的公共卫生线索。β-内酰胺类抗生素是最常用的抗菌药物，其目的是抑制细胞壁的合成。然而，新型β-内酰胺类药物的不断发展导致了数百种被称为β-内酰胺酶的拦截蛋白的出现和突变进化，这些蛋白赋予了对这些药物的耐药性。这些蛋白质通过氢键(H键)介导的化学机制使β-内酰胺类抗生素失活：形成β-内酰胺酰基酶酯并随后进行水解。直到最近，才有研究表明，氢键施加了巨大的电场，可以提供主要的动力，使酶反应速度提高5个数量级。预计在β-内酰胺酶中也会出现类似的情况，其中非常强的氢键，例如活性部位中的β-内酰胺和氧阴离子空穴之间的氢键，可能是唯一的动力，从而调节对抗生素的耐药性。有趣的是，β-内酰胺有一个固有的所谓斯塔克报告基团，即β-内酰胺羰基，它可以利用振动斯塔克效应来确定电场。利用生物学、振动光谱学和理论相结合的跨学科方法，拟议的项目旨在跟踪β-内酰胺酶活性部位内电场的演变，沿着临床记录的突变株导致抗生素耐药性的出现。具体地说，这项工作将集中在进化途径TEM1-TEM12-TEM10-TEM5(CAZ-1)上，该进化途径导致已经能降解第三代头孢菌素等抗生素的超广谱β-内酰胺酶(ESBL)。从这个项目中获得的知识具有最高的生物医学相关性，可以帮助解决抗菌素耐药性的全球线索。

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.