Theoretical Study On Degradation Of Typical Halogenated Hydrocarbon Pollutants In Enzyme Systems

Halogenated hydrocarbon products have become indispensable materials in agriculture,medicine,industry and life.They not only facilitate our daily lives,but also play an irreplaceable role in economic growth.However,while enjoying its benefits,we must also see that many products of halogenated aliphatic hydrocarbons and halogenated aromatic hydrocarbon are currently the main persistent organic pollutants(POPs).They have been subject to long-range word transport through various environmental media(water,soil,atmosphere,organisms,etc.),not only causing serious harm to the environment,but also causing various human health problems.At present,there are many methods for degrading halogenated alkane pollutants,including physical methods,chemical methods,biological methods,etc.And biological methods have the advantages of environment-friendly degradation and would not produce more poisonous by-products.The essence of biodegradation is to use enzymes in organisms(plants,animals,microorganisms,etc.)to reduce or completely eliminate the toxicity of pollutants.In experiments,certain specific microbial families have been determined to complete the dehalogenation of halogenated alkane pollutants.In addition,some enzymes responsible for degradation have also been successfully crystallized.However,the mechanism channels and the structural information of the intermediates in the degradation process have not been captured experimentally,which would hinder the further research and design of biological enzymes to catalyze the degradation of environmental pollutants.With the development of quantitative calculation and molecular simulation technology,computational simulation technology has been able to successfully overcome this problem,and using molecular dynamics(MD)technology and quantum mechanics/molecular mechanics(QM/MM)combined technology could achieve the simulation of biological enzymatic reactions under microscopic conditions.This dissertation selects two representative halogenated hydrocarbon pollutants and corresponding biological enzymes.One is the 1-chlorobutane(1-CB)and haloalkane dehalogenase LinB enzyme,and the other is the polychlorinated biphenyls(PCBs)and the dioxygenase BphC/hydrolase LigY enzymes.And we combined MD and QM/MM to study the dehalogenation process of 1-CB,and designed LinB modified enzymes with higher degradation efficiency based on mechanism analysis.And this work also studied the ring opening and chain scission mechanism of PCBs.This dissertation determined the detailed information of the enzymatic reaction of halogenated hydrocarbon pollutants,and supplemented the existing experimental results and theoretical explanations.In addition,it also deepened the understanding of biological enzymes and promoted the application of microbial degradation method in the environment.1.The effect of Histidine with different protonated states on the degradation mechanism of 1-CB1-CB is one of the representative pollutants in chlorinated aliphatic hydrocarbon materials,and clarifying its degradation mechanism can help understand the properties and degradation mechanism of other chlorinated aliphatic hydrocarbon pollutants.The degradation of 1-CB includes dechlorination and hydrolysis process.It is unclear whether the nitrogen atom in the imidazole group of Histidine 107 closed to the catalytic water molecule carries hydrogen proton would affect the hydrolysis process,and the current crystallization technology has not been able to determine accurately the position of the hydrogen atom.Therefore,determining the protonation state of the Histidine 107 is the prerequisite to figure out the degradation mechanism of 1-CB.This dissertation used MD and QM/MM to simulate the effects of different protonation states of Histidine 107 on the dechlorination process and hydrolysis process of 1-CB.It was found that there is no weak interaction between the water and the Histidine 107 when the proton was located at the nitrogen near from the water,which could make it easier to initiate the dechlorination and hydrolysis reaction.This dissertation not only clarified the dechlorination and hydrolysis mechanism of 1-CB,but also provided guidance for the subsequent design of modified enzymes.2.Research on the catalytic degradation of 1-CB by the theoretically designed LinB modified enzymeModified enzymes play a vital role in the study of enzymatic reactions.They could effectively improve the catalytic function of enzymes,and even catalyze some non-reactive substrates.Research on theoretically design of modified enzymes can help broaden the way of microbial degradation of environmental pollutants.In the degradation of 1-CB,experiments have pointed out that Leucine 248 is a mutatable amino acid in the active region of LinB enzyme,which is located between the substrate and Histidine 272.And more importantly,its side-chain can play the role of separating the active site from the cavity in LinB protein.Therefore,it is uncertain whether Leucine 248 is mutated to other amino acids with larger or smaller side-chain could improve the reactivity,which is the highlight.In this dissertation,based on experimental data,two nonpolar residues with different side-chain(Phenylalanine and Alanine)were selected to investigate the impact on the degradation activity.The MD and QM/MM methods were used to study the degradation efficiency of LinB modified enzyme to 1-CB.It was found when the residue Leucine 248 was mutated to phenylalanine with a larger side-chain,the active area was enlarged,and the phenyl ring of phenylalanine blocked the active region and hindered interactions between the substrate and other residues in the MM region,assisting the elimination of chloride ion from 1-CB.In addition,the energy barrier required in the dechlorination and hydrolysis process dropped significantly.Our quantitative calculation simulation could supplement and confirm the experimental conjecture,and provide theoretical guidance for the design of novel LinB modified enzymes.3.Catalytic mechanism for 2,3-dihydroxybiphenyl ring cleavage by nonheme extradiol dioxygenasesPolychlorinated biphenyls(PCBs)are one of the most representative pollutants of chlorinated aromatic hydrocarbons,and the most typical persistent organic pollutants in the Stockholm Convention list.They can accumulate in human organs and cause organ damage and exhaustion,and even cause irreversible damage to the nervous system.As biphenyl is the primary substrate for these PCBs/biphenyl degradation bacteria,the gene clusters responsible for the PCB/biphenyl aerobic degradation are named as bph(including BphA,BphB,Bphc and BphD).They can completely decompose PCBs into chlorobenzoic acid and 2-hydroxy-2,4-dienoic acid.And the dioxygenase BphC containing metallic iron can complete the ring opening of 2,3-dihydroxy polychlorinated biphenyl and could subsequently convert it into the 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate(HOPDA).In addition,the BphC enzyme can eliminate the toxicity of biphenyl and this ring-open degradation is the most important step in the entire degradation process.Therefore,figuring out its ring-opening mechanism will help deepen the understanding of PCBs degradation.In this dissertation,taking 2,3-dihydroxybiphenyl for instance,the MD and QM/MM method were used to explore the oxygen addition ring-opening degradation mechanism of biphenyl in the multi-state(triplet,quintet,and septet)system.The septet state was found to be the initial state when the dioxygen group entered,and then the quintet was found to be the ground reactive state for the subsequent catalytic reactions.The attack of the dioxygen group on DHBP consists of two steps.The first step is a proton-coupled electron transfer(PCET)step from DHBP to Histidine 194,where Histidine 194 acts as an acid-base catalyst.In the second step,the proton is transferred from the protonated Histidine 194 to the proximal oxygen of the superoxide ligand,and the HOO-species is determined to be the reactive oxygen species for subsequent attack.In addition,this dissertation also found that Criegee rearrangement occurs during ring opening of 2,3-dihydroxybiphenyl,and Histidine 194 can play an important role as a proton donor in the final reaction.This dissertation could provide a clearer theoretical basis for the degradation mechanism of PCBs by dioxygenase BphC,and also provide guidance for similar theoretical research.4.Study on the mechanism of C-C fission by meta-cleavage product hydrolase LigYThe pollutants PCBs undergo ring-opening degradation by BphC oxidase to produce meta-cleavage product(MCP)HOPDA,and subsequently MCP C-C bond fission by BphD hydrolase in the last step of the degradation process.BphD hydrolase could use the catalytic triad(Ser112-His265-Glu237)to complete the catalytic acylation reaction.In this dissertation,an amidohydrolase named LigY(Sphingobium sp.SYK-6)could also catalyze the MCP C-C bond fission.Interestingly,LigY is the only member of the amidohydrolase superfamily that has the capability to catalyze the MCP C-C bond fragmentation.LigY is a hydrolase containing metal zinc,and significantly the coordination number of a zinc ion and the binding mode between the zinc ion and the substrate(monodentate or bidentate)might have a crucial role to start the whole reaction.Therefore,in this dissertation,taking 4-carboxyl HOPDA as an example,the MD method and QM/MM method are used to simulate the degradation mechanism of HOPDA under the LigY hydrolase.It was found that the reaction was easier to occur in a monodentate mode and five-coordination status.And Tyrosine 190 could act as a proton donor to the deprotonated water required and then activate the catalysis.In addition,to gain further detailed observation of the tautomerization,the electrostatic potential on the molecular vdW surface was drawn in this dissertation.The data showed that Tyrosine 190 could also act as a proton transfer bridge to start the reaction in the bidentate mode and five-coordination status.This dissertation not only broadens the research on the degradation mechanism of MCP products,but also expands the research on the gene pool of biodegradable MCP products.