| One of the mechanisms of super bacterial resistance is the production of metallo-beta lactamase?M?L?to hydrolyze antibiotics.The FEZ-1 enzyme from Legionella is a member of the B3 subgroup of M?L,a double zinc that hydrolyzes a variety of antibiotics.However,at present,there are few studies on molecular recognition and interaction between the FEZ-1 enzyme and each antibiotic.This study selected FEZ-1 enzyme and cephalosporin antibiotics,cephthathione?CEF?,cephaloridine?LOR?and ceftazidime?CAZ?as the research objects,using synchronous fluorescence spectroscopy,molecular docking and dynamics simulation to study the changes of the conformation of enzyme molecules and the spatial distribution of antibiotic molecules during the binding process.The effects of different side chains of antibiotics on the hydrolysis ability of FEZ-1 were also analyzed to better design new antibiotics and provide a reference in the future.This topic mainly includes the following four aspects of research:?1?The FEZ-1 enzyme was expressed by genetically recombinant cloning and purified by dialysis to obtain a higher purity FEZ-1 enzyme.The protein concentration was determined to be 6.0×10-6mol/L.?2?The interaction between the FEZ-1 enzyme and the antibiotic molecule and the conformation of the enzyme molecule during the binding process were investigated by fluorescencequenchingspectrum,synchronousfluorescencespectroscopyand three-dimensional fluorescence spectroscopy.The results demonstrated that the three antibiotics and enzyme complex systems took place fluorescence quenching,indicating that all three cephalosporin antibiotics could interact with FEZ-1 enzyme by static quenching.The peak position of the synchronous fluorescence spectrum was red-shifted to different extents,CEF>LOR>CAZ,indicating that the microenvironment of the FEZ-1enzyme molecule changes and the hydrophily increases,further changing the conformation of fez-1 enzyme.?3?Molecular docking and dynamics simulation were used to research the changes of FEZ-1 enzyme molecular conformation during the binding of FEZ-1 enzyme to antibiotic molecules.These results suggested that CEF combined with FEZ-1 enzyme had the strongest binding force and lowest binding energy.The results of dynamics simulation indicated that the conformational changes of CEF and FEZ-1 system were most beneficial to the combination of them,that of CAZ and FEZ-1 system was more difficult.Moreover,the carbonyl carbon atoms on the parent ring of the CAZ molecule in the active pocket were deflected significantly and wound around the outer side of the active pocket,making it unable to bind well with the enzyme.?4?The results of binding free energy revealed that the free energy between FEZ-CEF and FEZ-LOR were lower,while the free energy of FEZ-CAZ was higher,which electrostatic and polar solvated were mainly driven force in all systems.The results of binding free energy decomposition showed that Zn264 has the largest contribution to the total binding free energy of the system,which is mainly in the form of electrostatic force.Conformational changes and energy analysis results demonstrated that it is harder to CAZ combined with FEZ-1 enzyme stability,which may be ascribe to the complex structure of the CAZ side chain,which forms a large steric hindrance in the active pocket of the enzyme molecule,resulting in difficulty in binding.It is supposed that side chain might weaken the interaction between the antibiotics mother ring on the carboxyl oxygen atoms and Zn264. |
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