Study On Degradation And Metabolism Activation Mechanism Of Typical Organochlorinated Pollutants Under The Action Of Biological Enzymes

Organochlorine pollutants are widely present in various environmental media,and their presence can be detected in air,water,soil,sediment and biological samples.Organochlorine pollutants come from chemical wastewater,the production and use of pesticides,the use of industrial materials,garbage incineration,and by-products of chlorine disinfection.Organochlorine pollutants can cause environmental pollution,aggravate human health risks,and endanger the diversity of wild animal and plant populations.There are many methods to study the degradation and metabolism of organochlorine pollutants,including physical,chemical and biological methods.Among them,the biological method is to extract enzymes from different strains to solve the severe environmental pollution problem.Understanding the metabolic transformation mechanism of enzyme-catalyzed exogenous pollutants in organisms is of great significance for evaluating the transformation fate,cleaner production,toxicological effects and health risks of environmental pollutants.There are many ways to study enzymes and enzyme-catalyzed reactions,which can be divided into experimental research and computational simulation research.Computational simulation based on quantum chemistry methods,that can study the reaction mechanism from a microscopic point of view,and explore the conversion pathway and product distribution of environmental pollution with enzymes.It can provide the details of the transition states and short-lived intermediates,of which the information is inaccessible by the experimental enzyme chemistry.Molecular dynamics(MD)and quantum mechanics/molecular mechanics(QM/MM)method have increasingly become a powerful tool to complement experimental enzyme chemistry.The MD,density functional theory(DFT)and QM/MM methods are used in this dissertation to study the biological fate of typical chlorinated organic pollutants.Predictive analysis of their degradation and metabolism mechanisms were carried out through computer simulation.The role of key amino acids in enzymes was discussed,and the combined use of Rosetta software provided a theoretical basis for the design of high-efficiency enzyme variants.Through the microscopic study of the enzyme-catalyzed reaction mechanism,the results of the experimental method were supplemented and verified.The present dissertation can improve the understanding of enzyme-catalyzed reactions,and promote its application in the environmental field.The detailed research contents are as follows:1.QM/MM Study of the Reaction Mechanism of Cl-cis,cis-muconate with Muconate Lactonizing EnzymeChloromuconic acid is one of the important toxic intermediate products in the degradation process of chlorobenzene.In this dissertation,we investigated the lactonization process of Cl-cis,cis-muconate with anti-MLE at the atomic level with the aid of a combined QM/MM approach.In the Cl-cis,cis-muconate system,two elementary steps were involved in lactanization process with four substrates.Our results provide the explicit structures of the enolate anion intermediates.Combined with DFT calculations,the energy barriers of catalytic reactions in non-enzymatic and enzymatic environments were compared,and the efficiency and specificity of enzymatic reactions were verified.The electrostatic influence analysis highlighted residues Arg51 and Gln294 for the 3-cis,cis-chloromuconic acid and the residue Asn193 for the 2-cis,cis-chloromuconic acid as the possible mutation targets for rational design of anti-MLE in future enzyme modification.The degradation mechanism of Cl-cis,cis-muconate was clarified,and the degradation pathway of chlorobenzene and its derivatives was perfected,which provided a theoretical reference for the design of highly efficient enzyme variats.2.Degradation Mechanism of Biphenyl and 4,4'-dichlorobiphenyl cis-dihydroxylation by Non-heme 2,3 Dioxygenases BphA:A QM/MM ApproachBiphenyl 2,3-dioxygenase(BphA),a Rieske-type and first enzyme in the aerobic degradation process,plays a key role in the metabolizing process of biphenyl/polychlorinated biphenyl aromatic pollutants in the environment.To elucidate the catalytic mechanism of BphA,we implemented QM/MM method to investigate the chemical transformations leading to the cis-diols.A hydroperoxo-iron(?)species is involved in the enzyme-catalyzed reaction.Herein,we explored the direct reaction mechanism of hydroperoxo-iron(?)species with biphenyl and 4-4'-dichlorobiphenyl.The dioxygenation process of the two substrates consists of three elementary reactions.The important roles of several residues during the dioxygenation process were highlighted via amino acid electrostatic effect and Rosetta amino acid tolerance sequence analysis.The results suggested that the amino acids Phe227,Val287,His323,Leu333,Ile336,Arg340 and Thr341 can be modified by mutations.This study supplemented and perfected the degradation mechanism of biphenyl compounds,and may provide theoretical support for further directed mutations and enzymatic engineering of BphA,as well as promote the development of degrading environmentally persistent biphenyl/polychlorinated biphenyl aromatic contaminants.3.Metabolic Activation Mechanism of 2,2',3,3',6,6'-hexachlorobiphenyl(PCB136)by Cytochrome P450 2B6:a QM/MM ApproachCytochrome P450 enzymes(CYPs)play an essential role in the bio-transformation of polychlorinated biphenyls(PCBs).The present work implemented quantum mechanic/molecular mechanic methods(QM/MM)and density functional theory(DFT)to study the metabolic activation of 2,2',3,3',6,6'-hexachlorobiphenyl(PCB136)catalyzed by CYP2B6.PCB136 was firstly catalyzed and converted by Compound I,and the electrophilic addition at the C? and C? sites would generate different reactive intermediates,ketone intermediates or epoxidized intermediates.The ? electrophilic addition reaction on the carbon atom of the benzene ring is the rate-determining step of the reaction,and the electrophilic addition energy barrier of C? is 10.9 kcal/mol higher than that of C?.C? position is the preferred site for the electrophilic addition reaction.Based on the previous experimental studies,this work investigated the mechanism of converting active intermediates into OH-PCB136,which has high toxicity in a non-enzymatic environment.Structural analysis via the electrostatic and noncovalent interactions indicates that ten residues play crucial roles in substrate recognition and metabolism.Among them,the amino acid residue Va1367 has a significant inhibitory effect on the electrophilic addition.The analysis confirmed that the halogen-?interactions are important factors for the metabolism of CYP2B6 to halogenated environmental pollutants,which is consistent with experimental research.This work improved the understanding of the metabolism and activation process of chiral PCBs,and point out that the introduction of gene mutations to reduce the higher deformation energy in the reaction can be used to guide the study of microbial degradation of PCBs.4.Study on Diclofenac Catalyzed by P450 Enzyme Active CenterAs an emerging environmental pollutant,diclofenac(DCF)has brought about an urgent environmental pollution problem and has become a topic of concern to environmentalists around the world.In this dissertation,MD simulation,amino acid interaction analysis and protein virtual mutation design were used to study the mutation candidate residues that improve the degradation efficiency of CYP105D7 on DCF.By calculating the free energy change of the protein-ligand complex after amino acid substitution,the contribution of the interaction between the residues to the stability of the complex is evaluated,and the stability of the complex also determines the degradation efficiency of DCF.Five structural flexible regions were determined by MD simulation,and the sequence numbers of perturbed residues were selected through analysis of amino acid interaction network(RIN)structure and centrality,combined with Rosetta software to determine the mutation site and calculate the free energy change between the mutant and the wild-type enzyme.Among the 270 mutants,25 mutation candidates with obvious enhancement effects were selected.It provides a theoretical basis for improving the degradation efficiency of CYP105D7 on DCF,and provides candidate residues for molecular modification experiments.