| Part 1 The structure characteristics of laccase from Tramemetes versicolor and the establishment of molecular docking modelObjectives: Based on the co-crystallized structure of laccase from Trametes versicolor(PDB code: 1KYA),the model of laccase-mediator system(LMS)was built to investigate the laccase-mediator interaction mechanism on molecular level.Methods: Based on the co-crystallized structure of 1KYA,the structural features of laccase's active site and the substrate's binding mode were studied using graphic display function of MOE.The model of laccase-substrate complex structures were built and the docking parameter settings were confirmed by using the molecular dock function of MOE.Results:1 At laccase's active site,hydrophobic amino acids Phe162,Leu164,Asn264,Phe265,Pro391,Gly392,Ala393,etc,could interact with substracts by hydropobic forces.The acidic amino acid Asp206 is the accepter of proton,and the basic amino acid His458 is the accepter of electron.2 The complex model was built by molecular docking,and the best docking parameter settings were confirmed.Use the docking parameter settings above,when we dock 2,5-xylidine,the original ligand of 1KYA,to the active site,the results show that the 2,5-xylidine superposed well with the ligand of co-crystallized structure,the parameter settings are as follows: Force field: MMFF94 x,Placement: Triangle Mather,Rescoring: London d G,Refinement: Forcefield.Part 2 Based on molecular docking to investigate the binding mode and the interaction mechanism between laccase and proton transfer mediatorObjectives: In order to discuss and reveal the interaction relationship of laccase and proton transfer mediator in laccase-mediator system,this study will based on the molecular docking to simulate the binding mode of mediator and from Trametes versicolor,the mechanism of laccase and mediators was explained on molecular level.Methods: Based on molecular docking,the model of laccase-mediator complex structure was built to revel the structural features and molecular interaction mechanism with laccase on proton transfer mediator,providing the direction for mediator's structural modification.Results: In the interaction between laccase and proton transfer mediator,the Asp206 hydrogen bonded with the-OH of the mediator,then,the mediator turn into nitroxide radical or phenoxyl radical;Part 3 Based on molecular docking to investigate the binding mode and the interaction mechanism between laccase and electron transfer mediatorObjectives: In order to discuss and reveal the interaction relationship of laccase and electron transfer mediator in laccase-mediator system,this study will based on the molecular docking to simulate the binding mode of mediator and from Trametes versicolor,the mechanism of laccase and mediators was explained on molecular level.Methods: Based on molecular docking,the model of laccase-mediator complex structure was built to revel the molecular interaction mechanism with laccase on electron transfer mediator,providing the direction for mediator's structural modification.Results: In the interaction between laccase and electron transfer mediator,His458 is the accepter of the electron,after the electron transfer,the mediator turn into the cation radical.Part 4 The molecular mechanism study of laccase-mediator--organophosphate poison ternary complexObjectives: Based on molecular simulation to investigate the interaction mechanism of laccase-mediator-organophosphate poison ternary complex(LMO).Methods: The complex model of LMO was built by using molecular docking,the binding mode and the interaction mechanism of ternary complex were reveled;In the LMO the conformation change of acceptor's C? was simulated by using molecular dynamics simulation.Then,the stability of ternary complex and the interaction mechanism of LMO were reveled.Results: In the LMO model,the binding conformation indicates that the mediator at the active site of the laccase interacts with the Asp206 via hydrogen bond,and interacts with neutral amino acids via hydrophobic interaction and ?-? accumulation.With the interactions,the mediator translates into free radical,then contacts with the(Organophosphate Poisons)OPs at the edge of the laccase's active site.The results of molecular dynamics simulation show that when the ternary complex reach a steady conformation,the His458 at the active site closes to mediator and stands off the type?copper ions,this promote the electron transfer between laccase and mediator,and leads to ?lack of electron? property of T1 Cu,then,the oxidation potential and the efficiecy of electron transfer increase.Part 5 Construction of the database with basic unit molecular of lignin degradation productsObjectives: More small molecule compounds with basic structural units of lignin degradation products were collected by literature search,the database with basic unit molecular of lignin degradation products was built to explore new mediators.Methods: Though literature review,moleculars which have the structural features of lignin degradations products were summarized,and using the module of MOE-Database,Molecular with Basic unit of Lignin degradation products Database(MBLD)was built.Results: Through literature review,we obtained 27 compounds with basic unit of lignin degradation products,and the MBLD was built,the database involves 2D and 3D structural information,provides the foundation for database screening.Part 6 Based on molecular docking and molecular similarity search to screen new mediatorObjectives: Through the MBLD and TCMD screen,to find new laccase mediators.Methods: By using methods of similarity searching and molecular docking to filter the MBLD and the TCMD,then potential laccase mediator will be obtained by structural modification and theoretical verification of these lead compounds.Results: Based on similarity searching and molecular docking,to search the MBLD and the TCMD,after the virtual screening and analyzing the binding mode,26 compounds were priority to pick out.These compounds are similar to natural mediator in structural features,differences with natural mediator is mainly reflects in the type and location of the substituent groups.Part 7 The structural modification of laccase mediator and theoretical verificationObjectives: Syringic alcohol and methyl syringate were used as templates,by structural modification of ortho substituent the best structural modification scheme was determined.Methods: Based on molecular docking,dock the new molecular to laccase active site.Results: Using molecular model and molecular docking to investigate the binding mode and reactivity between mediator and laccase.Computational simulation results show that the ortho-substituting groups of mediator's phenolic hydroxyl could maintain the stabillty of conformation during the interaction with laccase,besides,the electron donating capability of ortho-sbustituting groups could improve the stability of phenoxy radical;para-substituting groups' electron withdrawing capability of mediator's phenolic hydroxyl could strengthen the stability of ?-? stacking between mediator and Phe265 at laccase active site,but the strong electron withdrawing capability of para-substituting groups may be unfavorable to improve the reactivity of mediator and the stability of phenoxy radical.Part 8 Theoretical study of new mediator for degrading organophosphate poison in laccase mediator systemObjectives: Use syringetin,dehydrodisoeugenol and its reformed compounds in LMS to degrade organophosphate poison,to verify the possibility of new compounds to become laccase mediator.Methods: Based on molecular dynamics simulation to verify the effect of new compounds on degradation of organophosphate poison.Results: From the compounds after virtual screening,we select dehydrodiisoeugenol and syringetin as objects.The structural modification is based on mechanism study results and chemical structure theory,the ortho-groups of phenolic hydroxyl were replaced by two allyl acid substituenes.When used in the LMO model,the new compounds interact with laccase,the type and amount of interaction force increased,the combination with the laccase is more stable,the binding free energy is lower.Conclusions:1 In the process of interaction between laccase and mediator,neutral amino acids at the active site help stabilize the conformation of mediator,Asp206 is the accepter of proton and His458 is the accepter of electron,2 Computational simulation results show that the ortho-groups of mediator's phenolic hydroxyl play an important role on binding mode,besides,the electron donating capability of ortho-substituting groups could improve the stability of phenoxy radical;para-substituting group's electron withdrawing capability could strengthen the stability of ?-? stacking between mediator and Phe265 at laccase active site,but the strong electron withdrawing capability of para-substituting group may be unfavorable to improve the reactivity of mediator and the stability of phenoxy radical.3 In LMO,mediator binds at the laccase's active site,the OPs bind at the edge of active site,the OPs degrade via mediator free radical.When the LMO reach a steady conformation,laccase's conformation mutants.This conformation change improves the potential of laccase,promotes the electron transfer between laccase and mediator,also contribute to the degradation of OPs.4 By virtual screening to the MBLD and the TCMD,26 lead compounds were priority to pick out,taking the binding mode,molecular structural features,binding free energy into consideration,we obtained the compounds Syringetin and Dehydrodiisoeugenol which have the potential to become mediator.5 The results of natural mediator's structural modification show that when allyl acid substituene as the ortho-substituting groups,it contribute to a more stable conformation binding mode,and avoid the formation of intra-molecular hydrogen bonding.6 After the structural modification of Syringetin and Dehydrodiisoeugenol,the catalytic efficiency of new compounds increases,and the binding mode is more stable,the rationality of structural modification is verified. |
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