Mechanism Study On The Interaction Of Metal β-lactamases With β-lactam Antibiotics Based On Spectroscopic Methods And Computional Simulations

In recent years,with the widespread use of antibiotics,bacterial resistance is becoming increasingly prominent,metallo-β-lactamase(MβL)is important as a major source of resistance of pathogenic bacteria to the widely used β-lactam antibiotics.Therefore,the study on MβL is the key to solve this problem.To achieve this lofty goal,we must first understand the essential question of “how MβL and β-lactam antibiotics interact at the atomic level”.It is difficult to obtain the crystal structure of the MβL-substrate complex,thus the above issue cannot be solved by experiment.In this study,in order to give a reasonable answer to this issue,the molecular recognition and interaction of MβL(Bc II,CphA,and FEZ)with β-lactam antibiotics(PV,CFX,IMP,and Azt)in the binding process were studied through spectroscopy analysis,molecular docking and molecular dynamics simulation.This study may provide some valuable reference and inspiration for the development of new antibiotics to resist the hydrolysis of MβL.The main contents of this paper are as follows:1.CphA and FEZ were expressed in E.coli BL21(DE3)cells containing pET28b-CphA and pET28b-FEZ,along with the purchased Bc II,three representative MβLs were finally obtained.Under conditions of low temperature and excess substrate,the interaction between MβLs and bicyclic β-lactam antibiotics(PV,CFX and IMP)was analyzed using synchronous fluorescence and three-dimensional fluorescence spectroscopy.The results indicated that the binding of the substrate can cause the conformational change of MβL,suggesting that there is an induced-fit process between the substrate and MβL during the binding process.This induced-fit effect in a order from strong to weak is Bc II,FEZ and CphA.2.The molecular recognition and interaction of MβL with bicyclic β-lactam antibiotics(PV,CFX and IMP)and their hydrolysates were further investigated by molecular dynamics simulation.The results showed that: Unlike single zinc CphA,both substrate and product have similar binding mode with bis-zinc MβL(Bc II and FEZ).Electrostatic interaction between zinc ion of MβL binding pocket and carboxyl group ofβ-lactam antibiotics provide main driving force for the binding process.Bc II and FEZ have flexible binding pockets,especially Bc II,allowing the β-lactam antibiotics and MβL to adapt to each other through induced-fit mechanism.Being abducted by substrates,MβL canmatch perfectly with different antibiotics by changing the conformation of the loops near the binding pocket.On the contrary,the binding pocket of CphA is less flexible that leads to a weaker induced-fit effect for the binding process.3.The interaction mechanism between MβL and Azt(monocyclic β-lactam antibiotic)was investigated by means of spectroscopic and molecular docking methods.The results indicated that the binding affinity between Azt and MβL was weak.The main reason is that the sulfonic acid group in Azt greatly attenuates the electrostatic interaction of with MβL,and it leads directly to the weak binding affinity between Azt and MβL,suggesting that the monocyclic β-lactam nucleus can be used as a novel antibiotic backbone molecule against the hydrolysis of MβL.4.Taking E.coli BL21(DE3)cells containing pET28b-CphA as the experimental object,the evolution and migration of the recombinant bacteria in Biapenem(BIA)medium were investigated.The results showed that the bacteria rapidly enter dormancy period to avoid antibiotic attack when they contact lethal dose of BIA,and then the bacteria began to accumulate drug-resistance mutations that allowed the bacteria to tolerate lethal doses of BIA and continue to grow and migrate.Under the pressure of Biapene selection,CphA enzyme ultimately evolved into a more efficient enzyme through genetic mutations.The same experimental scheme was implemented to explore the variation and evolution of the recombinant strain in Azt medium.However,no growth or migration of the recombinant strain in Azt was observed.5.we constructed MβL chromatographic models,and discussed their application potential on bioactive compounds screening from traditional Chinese medicine.In summary,the recognition of bicyclic β-lactam antibiotics by MβL is mainly achieved through the bicyclic β-lactam nucleus in antibiotics molecule.Electrostatic interaction between zinc ion of MβL binding pocket and carboxyl group of β-lactam antibiotics provide main driving force for the binding process.Without changing bicyclic β-lactam nucleus,traditional strategy of developing newly functionalized groups may be readily thwarted by the plastic nature of the MβL binding pocket(Induced-fit effect).In addition,MβL can become a more efficient enzyme through genetic mutations,and it also poses a threat to new improved bicyclic β-lactam antibiotics.However,the monocyclic β-lactam antibiotics have no suchshortcomings.Therefore,we could develop more novel MβL-resistant β-lactams based on the ideal scaffold of the monocyclic β-lactam nucleus.