Molecular Recognition And Binding Between Bacteroides Fragilis CcrA Enzyme And Cephalosporin Antibiotics

The presence of super-bacteria makes most of the ?-lactam antibiotics used clinically ineffective,mainly because it can produce metallo ?-lactamase,which can hydrolyze antibiotics.Metallo ?-lactamase CcrA from acteroides fragilis is a typical B1 subgroup enzyme,however,few studies on the binding process between metallo ?-lactamase CcrA and antibiotics are done.In this study,the molecular recognition and interaction mechanism between metallo p-lactamase CcrA and cefotaxime(CTX)and ceftazidime(CAZ)were studied by means of experimental determination and theoretical prediction.The main contents are as follows:1.The metallo ?-lactamase CcrA was expressed in E.coli BL21(DE3)and was purified by nickel ion affinity chromatography,then identified by SDS-PAGE and finally determined the protein yield by Bradford.The results showed the process achieved efficient expression of the target protein CcrA,the purity of CcrA enzyme was higher after purification,and the yield of CcrA enzyme was 0.26-0.28 mg/mL.2.Molecular recognition and interaction between CcrA enzyme and two cephalosporins antibiotics(CTX and CAZ)were studied by fluorescence spectroscopy.The fluorescence spectra of antibiotics and CcrA enzyme were measured at three temperatures(277K,281K,285K).Fluorescence quenching constant Ksv and quenching rate constant Kq,binding constant Ka,binding site number n,the thermodynamic parameters were obtained.The experimental results showed that the binding process between the two antibiotics and CcrA enzyme were static quenching.At the same temperature,the Ksv and Kq of CcrA-CTX binding system were both higher than that of CcrA-CAZ binding system;the binding constant Ka and the binding site number n of CcrA-CTX binding system were also larger.This indicated that the affinity of CcrA to CTX and the stability of complex CcrA-CTX were higher than that of CcrA-CAZ binding system.The experimental results indicated that the binding process between CcrA enzyme and two antibiotics were spontaneous exothermic process because of the negative enthalpy and negative Gibbs free energy.3.Molecular recognition and interaction between CcrA enzyme and two cephalosporins antibiotics(CTX and CAZ)were studied by molecular docking simulation,which could show the interaction patterns of amino acid residues and small molecule ligands.The results showed that the loop near the active pocket of the enzyme were highly flexible,indicating that the antibiotic entered into the enzyme active pockets.The flexibility of the loop is more favorable for the binding of the enzyme to the antibiotic,which also corroborated the conclusion of the above experiment.The Gibbs free energy of the two binding systems obtained by the docking were negative,indicating that the binding process between the CcrA enzyme and the two antibiotics are spontaneous,which are consistent with the experimental results.