Molecular Recognition And Binding Between Aeromonas Hydrophila CphA Enzyme And Carbapenem Antibiotics

The metallo-beta-lactamase(M?Ls)prevents beneficial behaviors of P-lactam antibiotic against bacterial infections by hydrolyzing the ?-lactam ring.From the Aeromonas hydrophila CphA enzyme is a member of M?Ls subgroup,which has only one Zn2+ at the active site and specifically hydrolyzes carbapenem antibiotics.However,no more reseach on the molecular recognition and binding between CphA enzyme and carbapenem antibiotics are available.In this paper,on the basis of obtaining CphA enzyme by expressing in E.coli BL21(DE3),we researched its conformation changes,spatial distribution of antibiotics about imipenem(IMP)and biapenem(BIA),the essential of the complexes formed,the type of interaction and the number of sites during their binding process by fluorescence spectra and molecular docking.(1)The CphA enzyme was expressed in E.coli BL21(DE3)containing pET28b-CphA and purified by cation exchange chromatography,then identified by SDS-PAGE and determined protein yield by Braford,finally characterized by fluorescence spectra.The result showed that the molecular weight of CphA enzyme was about 28 KDa,the yield was about 0.18?0.21 mg/mL,and the fluorescence was significant at 336.2 nm when the excitation wavelength was 278 nm.(2)The Km of CphA with IMP and BIA were 356 ?M and 286 ?M at 277 K,respectively.The ratios of between them in saturation process by fluorescence emission spectroscopy were 0.95 and 0.98,respectively.The results of synchronous fluorescence spectra showed that they interacted and the micro-environment of Tyr residues changed slightly.The results under at 277 K,281 K and 285 K indicated that the binding process were static quenching,they interacted through only one type of binding site in the binding process,and they were spontaneous exothermic process from the thermodynamic parameters.(3)The results of molecular docking showed that there were some similarities between the CphA enzyme with IMP and BIA during the binding process:the C3-carboxyloxy of the two antibiotics ?-lactam rings directly formed a metal coordination bond with Zn2+,and the loop of the CphA undergone a local conformation changes.Due to smaller the side-chain of its,the whole IMP entered into the pockets,and there were 3 sets of electrostatic force and 5 groups of hydrogen bonds during the binding process;Part of BIA was left in the pocket outside due to larger bicyclic triazole of its side-chain,there were 4 sets of electrostatic forces and two groups of hydrogen bonds during the binding process,so the affinity of CphA with BIA was greater than that of IMP,and the stability of CphA-BIA complex was also greater than the CphA-IMP complex.The Ksv and Ka of the CphA-BIA complex system from the fluorescence spectrum results were higher than those of the CphA-IMP complex system,which also corroborated these above inferences.In addition,the docking results showed that the Gibbs free energy of the complex systems were negative,indicating that their binding processes were the spontaneous exothermic process,which were consistent with the previous thermodynamic results.