Abstract:
Metallo-β-lactamases (MβLs) hydrolyze and inactivate β-lactam antibiotics, are a pivotal mechanism conferring resistance against bacterial infections. SMB-1, a novel B3 subclass of MβLs from Serratia marcescens could deactivate almost all β-lactam antibiotics including ampicillin (AMP), which has posed a serious threat to public health. To illuminate the mechanism of recognition and interaction between SMB-1 and AMP, various fluorescence spectroscopy techniques and molecular dynamics simulation were employed. The results of quenching spectroscopy unraveled that AMP could make SMB-1 fluorescence quenching that mechanism was the static quenching; the synchronous and three-dimensional fluorescence spectra validated that the microenvironment and conformation of SMB-1 were altered after interaction with AMP. The molecular dynamics results demonstrated that the whole AMP enters the binding pocket of SMB-1, even though with a relatively bulky R1 side chain. Loop1 and loop2 in SMB-1 undergo significant fluctuations, and α2 (71-73) and local α5 (186-188) were turned into random coils, promoting zinc ion exposure consistent with circular dichroism spectroscopy results. The binding between them was driven by a combination of enthalpy and entropy changes, which was dominated by electrostatic force in agreement with the fluorescence observations. The present study brings structural insights and solid foundations for the design of new substrates for β-lactamases and the development of effective antibiotics that are resistant to superbugs.
摘要:
金属-β-内酰胺酶(MβLs)水解和灭活β-内酰胺抗生素,是赋予细菌感染抗性的关键机制。SMB-1是粘质沙雷氏菌MβLs的一种新型B3亚类,可以使几乎所有β-内酰胺抗生素(包括氨苄青霉素(AMP))失活。这对公众健康构成了严重威胁。为了阐明SMB-1和AMP之间的识别和相互作用的机制,采用各种荧光光谱技术和分子动力学模拟。猝灭光谱的结果揭示了AMP可以使SMB-1荧光猝灭,其机理为静态猝灭;同步和三维荧光光谱验证了SMB-1与AMP相互作用后的微环境和构象发生了变化。分子动力学结果表明,整个AMP进入SMB-1的结合袋,即使具有相对较大的R1侧链。SMB-1中的Loop1和Loop2发生显著波动,将α2(71-73)和局部α5(186-188)转化为无规卷曲,促进锌离子暴露与圆二色谱结果一致。它们之间的结合是由焓和熵的变化共同驱动的,与荧光观察结果一致,静电力占主导地位。本研究为设计β-内酰胺酶的新底物和开发对超级细菌具有抗性的有效抗生素提供了结构见解和坚实的基础。