Insights into the Enhanced Ceftazidime Hydrolysis by Ent385 AmpC β‑Lactamase from Multiscale Simulations

The emergence of multidrug-resistant bacteria poses a significant threat to public health. Particularly, they are becoming increasingly resistant to β-lactam antibiotics, which are one of the most important drug classes for the treatment of bacterial infections. Ceftazidime-avibactam has shown promising activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. However, an Ala294-Pro295 deletion in the Class CE. cloacaeAmpC β-lactamase can confer reduced susceptibility to these agents. In this study, we investigated the molecular mechanisms underlying the enhanced hydrolysis of ceftazidime by E. cloacae Ent385 AmpC β-lactamase with the deletion using quantum mechanics/molecular mechanics (QM/MM) simulations. We used constant pH molecular dynamics simulations of the β-lactamase-ceftazidime acyl-enzyme complex to verify the likely protonation states, confirming Tyr150 primarily exists as a tyrosinate. We then used QM/MM (DFTB2/ff14SB) umbrella sampling to calculate the reaction-free energy barriers (Δ‡G) for the deacylation step of cephalosporin hydrolysis. This reveals that Tyr150 (rather than the substrate) acts as the base. Importantly, the difference in Δ‡G between the canonical E. cloacae AmpC (P99) and the Ent385 variant with Ala294-Pro295 reinserted, on the one hand, and the Ent385 variant, on the other, was in very good agreement with the difference deduced from experimental kinetic data. Detailed analysis of the transition state ensembles, alongside additional simulations, shows that the Ala294-Pro295 deletion allows the entrance of an additional water molecule that helps stabilize the tetrahedral intermediate. Overall, our QM/MM simulations provide valuable insights into the reaction mechanism and reasons for enhanced ceftazidime breakdown. The protocol used in this study successfully captures the kinetic differences observed among the studied variants. This approach can be employed to investigate other Class C β-lactamase variants with similar features, providing insights into their mechanisms and potential contributions to reduced susceptibility to antibiotic treatments.多重耐药细菌的出现对公共卫生构成严重威胁。其中，它们对β-内酰胺类抗生素（β-lactam antibiotics）的耐药性不断增强，而这类抗生素是治疗细菌感染最重要的药物类别之一。头孢他啶-阿维巴坦（Ceftazidime-avibactam）对包括碳青霉烯类耐药肠杆菌目（carbapenem-resistant Enterobacterales）在内的高度耐药细菌显示出良好的抗菌活性。然而，阴沟肠杆菌C类AmpC β-内酰胺酶中的Ala294-Pro295缺失突变，会导致菌株对这类药物的敏感性降低。本研究中，我们通过量子力学/分子力学（quantum mechanics/molecular mechanics, QM/MM）模拟，探究了带有该缺失突变的阴沟肠杆菌Ent385 AmpC β-内酰胺酶增强头孢他啶水解作用的分子机制。我们利用β-内酰胺酶-头孢他啶酰基酶复合物的恒pH分子动力学模拟，验证了其可能的质子化状态，证实Tyr150主要以酪氨酸负离子形式存在。随后，我们采用QM/MM（DFTB2/ff14SB）伞形采样方法，计算了头孢菌素水解脱酰基步骤的反应自由能垒（Δ‡G）。结果显示，Tyr150（而非底物）充当了碱催化剂。值得注意的是，野生型阴沟肠杆菌AmpC（P99）与重新插入Ala294-Pro295的Ent385突变体，和单纯的Ent385突变体之间的Δ‡G差异，与实验动力学数据推导得到的差异高度吻合。对过渡态集合的详细分析以及额外模拟表明，Ala294-Pro295缺失允许额外水分子进入，从而稳定四面体中间体。总体而言，我们的QM/MM模拟为头孢他啶增强降解的反应机制及相关原因提供了宝贵见解。本研究使用的实验方案成功复现了所研究变体间的动力学差异。该方法可用于研究其他具有类似特征的C类β-内酰胺酶变体，为其耐药机制以及对抗生素治疗敏感性降低的潜在原因提供科学参考。