多光谱方法结合理论计算探究金属β-内酰胺酶SMB-1与亚胺培南之间的相互作用  

Structural Insight Into Interaction Between Imipenem and Metalβ-Lactamase SMB-1 by Spectroscopic Analysis and Molecular Docking

作　　者：张椰莉 程建伟 董晓婷 边六交[2] ZHANG Ye-li;CHENG Jian-wei;DONG Xiao-ting;BIAN Liu-jiao(Department of Biology,Taiyuan Normal University,Jinzhong 030619,China;College of Life Science,Northwest University,Xi’an 710069,China;Institute of Geographical Science,Taiyuan Normal University,Jinzhong 030619,China)

机构地区：[1]太原师范学院生物系,山西晋中030619 [2]西北大学生命科学学院,陕西西安710069 [3]太原师范学院地理科学学院,山西晋中030619

出　　处：《光谱学与光谱分析》2023年第7期2287-2293,共7页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(31971143);山西省基础研究计划项目(202103021223327)资助。

摘　　要：金属β-内酰胺酶(MβLs)可以水解几乎所有的β-内酰胺类抗生素,这是导致细菌感染治疗中产生耐药性的主要机制。迄今为止,由于缺乏临床批准的抑制剂,这已成为全球关注的问题。最近来自粘质沙雷氏菌的SMB-1被发现是一种新型的B3亚类MβL,它可以灭活几乎所有含β-内酰胺环的抗生素。为了明确SMB-1与β-内酰胺类抗生素的特异性分子识别和作用机制,采用内源性荧光光谱、同步荧光光谱、三维荧光光谱及分子对接方法对碳青霉烯类抗生素亚胺培南(IMIP)与金属β-内酰胺酶SMB-1之间的相互作用机制进行探究。猝灭光谱结果表明IMIP可以使SMB-1内源性荧光猝灭,且猝灭机制为动态和静态组合猝灭,其中静态猝灭为主,结合常数K a为16.11×10^(3)L·mol^(-1)(277 K),表明两者之间具有很强的结合力;根据Van’t Hoff方程得出结合过程中的热力学参数ΔG<0,ΔH=-79.65 kJ·mol^(-1),ΔS=-238.69 J·mol^(-1),说明两者的结合是由焓变和熵变共同驱动的,且氢键和范德华力为主要作用力;同步荧光结果中,随着IMIP浓度的增加,SMB-1最大发射波长均发生蓝移4.4和2.9 nm,表明Tyr和Trp残基参与IMIP与SMB-1的结合过程;三维荧光光谱中IMIP加入后,SMB-1的Peak B和Peak C强度显著降低,表明SMB-1与IMIP作用之后的微环境和构象发生了改变,与同步荧光结果一致。分子对接结果中IMIP的β-内酰胺环进入SMB-1的结合口袋,而侧链因空间位阻效应位于活性口袋的外部,表明SMB-1主要是识别IMIP的核心结构,与其R2侧链的作用较弱;与IMIP参与作用的氨基酸残基包括Ser175,Thr177,Gln157,His215和Glu217,表明这些氨基酸残基与活性部位的两个锌离子是设计具有强亲和力的SMB-1抑制剂的关键因素;结合自由能也为负值,表明两者的结合是一个自发放热的过程,与荧光结果一致。该研究提供了对SMB-1与IMIP的识别和结合的见解,可能有助于设计β-内酰胺酶新底物Metallo-β-lactamases(MβLs)could hydrolyze almost allβ-lactam antibiotics,the primary mechanism resulting in drug resistance against bacterial infections.This has become a substantial concern due to the lack of clinically approved inhibitors.SMB-1 from Serratia marcescents is a novel B3 subclass MβL that inactivates almost allβ-lactam-containing antibiotics.The interaction mechanism between carbapenem antibiotic imipenem(IMIP)and Metallo-β-lactamase SMB-1 was ascertained in this paper using endogenous fluorescence spectroscopy,synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy and molecular docking methods.The quenching spectrum results demonstrated that IMIP quenched endogenous fluorescence of SMB-1,and the quenching mechanism was a combination of dynamic and static quenching,of which static quenching is the core one;the binding constant Ka was 16.11×10^(3)L·mol^(-1)(277 K),indicating a strong binding force between them;the thermodynamic parameters in the binding process obtained from the Van’t Hoff equationΔG<0,ΔH=-79.65 kJ·mol^(-1),ΔS=-238.69 J·mol^(-1),illustrating that the binding was driven by both enthalpy and entropy changes and hydrogen bonding and van der Waals forces were the main forces;Moreover,the maximum emission wavelength of SMB-1 in synchronous fluorescence results was blue shifted by 4.4 and 2.9 nm with increasing IMIP concentration,revealing that Tyr and Trp residues were involved in both.The significant decrease of Peak B and Peak C intensity of SMB-1 with IMIP introduced in the three-dimensional fluorescence spectra indicated that the microenvironment and conformation of SMB-1 changed after the interaction with IMIP,which is consistent with the synchronous fluorescence results.Furthermore,theβ-lactam ring of IMIP entered the binding pocket of SMB-1 in the molecular docking results,while the side chain was located outside the active pocket due to the spatial site block effect,inferring that SMB-1 mainly recognized the core structure of IMIP and in

关 键 词：金属β-内酰胺酶SMB-1 亚胺培南 荧光光谱 分子对接 相互作用 

分 类 号：TS201.2[轻工技术与工程—食品科学]