The Molecular Mechanisms Study Of Hydrolytic Substrate And Interaction With Inhibitor Of Class B ?-lactamase Produced By Food-borne Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is a typical Gram-positive food-borne pathogenic microorganism widely existing in nature.In recent years,due to the overuse of antibiotics in the aquaculture industry,methicillin-resistant Staphylococcus aureus?MRSA?has been detected in livestock products and transmitted into the food chain,leading to food poison.With the increasing awareness of food safety and frequent outbreaks of food-borne Staphylococcus aureus infection,people pay more attention to the prevention and treatment of Staphylococcus aureus infection.MRSA secretes many kinds of drug-resistant enzymes,among which class B metallo-b-lactamases?MBLs?plays an important role in the drug resistance mediated by methicillin-resistant Staphylococcus aureus.They can hydrolyze almost allb-lactam antibiotics,and they are the main target of research and development of antimicrobial agents against methicillin-resistant Staphylococcus aureus.There are many documentations on MBLs,however,the molecular catalytic mechanism has not been clarified yet.Based on these facts,the catalytic hydrolysis mechanism of MBLs produced by several food-borne drug-resistant bacteria was analyzed and confirmed by molecular dynamics simulation,which provided a new idea for the development of new natural drug-resistant inhibitors and effective control of drug-resistant bacterial infections.The main results are as follows:1.Homology modeling of?-lactamase N1 structure and three-dimensional structure optimization of NDM-1 and VIM-1.The homologous template was determined by the analysis of the?-lactamase N1 amino acid sequence.Using the crystal structure of?-xylanase as template,the ULA-300 3D structure was constructed via Modeler module in Discovery Studio 2.5,and the 1000ns molecular dynamics simulation for the structure of ULA-300 was carried out.Finally,the stable 3D structure of ULA-300 was determined.The crystal structures of NDM-1?PDB code:3ZR9?and VIM-1?PDB code:5N5G?were used as the initial structure for the molecular modeling,and the molecular dynamics simulation of the 3D structures was carried out for 1000 ns.Finally,the stable 3D structures of NDM-1 and VIM-1 were determined.2.The hydrolysis mechanism of?-lactamase N1 to nitrocefinIn this paper,the complex system of?-lactamase N1 with nitrocefin was studied by molecular modeling and quantum mechanics/molecular mechanics?QM/MM?calculations.The catalytic mechanism of?-lactamase N1 for the hydrolysis of nitrocefin was predicted.Molecular dynamics simulations show that the catalytic reaction begins with the formation of hydrogen bonds between Gln171and water molecules,which is captured by?-lactamase N1 for catalytic hydrolysis.Subsequently,the carboxyl group coordinates with Zn₂ in the form of coordination bond.The binding energy decomposition showed thatPhe169 anchored nitro phenyl groups through the interaction between benzene rings and accumulation.Phe169 and Zn₂ locate nitrocarbene in a specific direction.The active sites of?-lactamase N1 are Gln171 andPhe169,which are essential for the hydrolysis of nitrocefin.3.The analysis of the interaction mechanisms of aztreonam with NDM-1 and VIM-1The specific binding modes of aztreonam with NDM-1 and VIM-1 were studied via molecular dynamics simulation.The binding energy calculation and molecular dynamics simulation results showed that aspartic acid?NDM-1:aspartic acid 124,VIM-1:aspartic acid 118?played a major role in the binding of protein with aztreonam.However,according to the binding free energy calculation,the binding energy between Asp118 and aztreonam is weaker than that of Asp124 with aztreonam.Through the analysis of the simulated trajectory,it is confirmed that in the complex system of VIM-1 and aztreonam,due to the interaction between His201,His240 side chains and thiazole ring,the binding affinity of aztreonam with Asp118decreased,resulting in the loss of hydrolysis activity of VIM-1 to aztreonam.4.The analysis of the interaction mechanisms of avibactam with NDM-1 and VIM-1Molecular dynamics simulations show that avibactam can bind to the active regions of NDM-1 and VIM-1.In NDM-1,residues of Ile35,Val73,His120,His122,Asp124,His189,Cys208,Lys211,Gly219,Asn220,His250 can form strong interaction with avibactam,and in VIM-1,Phe62,Tyr67,Trp87,His116,Asp117,Asp118,His179,Gly209,Asn210,His240 forms strong interaction with avibactam.In NDM-1-avibactam and VIM-1-avibactam complexes,avibactam can interact strongly with aspartic acid Asp124 and Asp118 in the active region(?35?E_total<-3 kcal/mol),resulting in the hydrolysis of avibactam by NDM-1 and VIM-1.These results indicate that Asp124 and Asp118 in NDM-1 and VIM-1 can effectively promote the hydrolysis of substrates.5.The inhibition mechanism analysis of theaflavins to NDM-1 and VIM-1The complexes of NDM-1 and VIM-1 binding with theaflavins were studied by the computational biology.The results showed that theaflavins could bind to the active regions of NDM-1 and VIM-1.They could bind to the active sites of NDM-1,such as Trp93,His122,Gln123,Asp124,Lys211,His250 and VIM-1,such as Tyr67,Asp118,His179,Val200,His201 and His240.The results of decomposition binding free energy showed that theaflavins could interact with amino acid residues in active region and form competitive binding with substrates in NDM-1-theaflavins and VIM-1-theaflavins complexes,leading to the loss of the hydrolysis activity of NDM-1 and VIM-1.