Time-resolved structural analysis of the extended spectrum beta-lactamase CTX-M-14

超广谱β-内酰胺酶CTX-M-14是目前临床上最常用的抗生素，它对β-内酰胺类抗生素具有耐药性，在细菌感染的治疗中发挥着重要作用。CTX-M-14 DT 12 β-内酰胺酶是从临床相关细菌肺炎克雷伯氏菌中分离的一种酶，具有扩展的底物谱（ESBL），不仅能够水解青霉素类，还能够水解头孢菌素类和单内酰胺类抗生素，因此导致对大量抗生素的耐药性。第一个目标是通过系列同步加速晶体学（SSX）确定稳定反应中间体（如Michaelis-Menten络合物、共价中间体以及产物络合物）的室温结构。这些静态的室温结构显示了与低温结构和时间分辨x射线衍射实验进行比较的重要基础。通过TR-SSX，我的目标是解决抗生素催化过程中的动态构象变化，到目前为止，这只能来自静态的低温结构。在最慢转换动力学的反应条件下，我将启动TR-SSX实验。使用之前建立的TR-SSX协议，例如通过将底物溶液液滴喷射到安装在芯片上的微晶上以引发反应，将能够以短时间间隔记录反应的快照。或者，光学笼状基底可用于反应引发。此外，我将利用最近建立的突发系列数据收集模式，它使许多时间点的收集在快速连续的时间分辨率为1.35毫秒。这些短的时间范围将使其有可能遵循结构中间体的底物在催化过程中。最后，这些实验将通过MD模拟进行补充，旨在连接构象变化的子步骤。

The aim of this proposal is to resolve the catalytic details of the reaction mechanism of the extended spectrum beta-lactamase CTX-M-14 with time-resolved serial synchrotron crystallography (TR-SSX).Beta-lactamase confer resistance to beta-lactam antibiotics, the most commonly prescribed antibiotics in the world, and thereby play an important role in the treatment of bacterial infections. The CTX-M-14 DT12 beta-lactamase is an isolate from the clinically relevant bacterium Klebsiella pneumoniae, which has an extended substrate spectrum (ESBL) and is able to hydrolyze not only penicillins but also cephalosporins and monobactam antibiotics and thus contributes to resistance against a large group of antibiotics. The first aim is to determine the room-temperature structures of stable reaction intermediates like the Michaelis-Menten complex, the covalent intermediate as well as the product complex, via serial synchrotron crystallography (SSX). These statis room-temperature structures display an important basis for the comparison to cryo-structures and time-resolved x-ray diffraction experiments. Via TR-SSX I aim to resolve resolve the dynamic conformational changes during catalysis of the antibiotics, which so far can only be derived from static cryo-structures. With the reaction conditions for the slowest turnover kinetics I will initiate TR-SSX experiments. Using the previously established TR-SSX protocols, e.g. by shooting droplets of substrate-solution onto the chip-mounted microcrystals for reaction initiation, will enable recording snapshots of the reaction at short time-intervals. Alternatively, optically caged substrates can be used for reaction initiation. Additionally, I will make use of a recently established burst-series data collection mode, which enables the collection of many timepoints in rapid succession at a time-resolution of 1.35 ms. These short timeframes will make it possible to follow structural intermediates of the substrate during the catalysis. Finally, these experiments will be complemented by MD-simulations, aiming to connect the substeps of the conformational changes.