Theorectial Study On The Catalytic Mechanism Of Fluoroacetate Dehalogenase

Halogenated compounds have the characteristics of good heat resistance,lipophilic,thermal conductivity,insulation,and biological activity.Thus they are produced in large quantities and widely used in production and life.However these compounds have caused serious environmental pollution and health problems owing to their direct toxicity,persistence and bioaccumulation.Biodegradation of dehalogenation is achieved by the effective enzymes that are produced by microbes,plants and animals.Different from traditional chemical methods,this technology is eco-friendly,which can be performed under mild conditions and does not require additional chemical input,so this technology plays a very important role in the degradation process of halogenated compounds.In recent years,based on the rapid development of computer technology,theoretical simulation technology has become a very important method in enzyme research.Through molecular dynamics simulation(MD)and the combination of molecular mechanics and quantum mechanics(QM/MM)method,the enzymatic reaction can be studied at the atomic level.The theoretical simulation can not only verify the reaction mechanism proposed in the experiment,but also can be used to guide the experiment to design the enzyme to improve the enzyme catalytic efficiency,saving time and economic cost of tranditional experiment.Compared with other dehalogenases,fluoroacetate dehalogenase(FAcD)has attracted wide attention because it has the ability to catalyze the strong C-F bond cleavage,whose dissociation energy is highest in nature products.In this paper,molecular dynamics simulation and the combination of molecular mechanics and quantum mechanics were used to investigate the reaction mechanism of FAcD catalyze defluorination.By analyzing the different structure parameters of reaction process,the correlation between the key structure parameters(bond length O?C?,bond angle O?C?F)and the energy barrier was found.This finding can be used to aid enzyme design to improve enzyme efficiency to achieve the purpose of degrading persistent PFCA.In addition,based on the analysis of amino acid electrostatic effects,Tyr68,Trp185,and Met215 were proposed as candidates for amino acid mutation research.On this basis,in order to provide a unified picture of the mechanism of FAcD catalytic dehalogenation,the mechanism of FAcD catalyze dechlorination and debromination was studied.According to QM/MM calculations,the energy barriers of FAcD for catalyze defluorination,dechlorination and debromination were 10.1,19.7 and 20.9 kcal mol-1,respectively,indicating that the efficiency of FAcD catalytic dehalogenation follows the following order:defluorination>dechlorination>debromination.By analyzing the structure,we confirmed that shorter length of Os-Ca and larger angle values of O?C?X is vital for the efficient cleavage,which is the same with defluorination reaction.The dehalogenation catalyzed order is explained by structure analysis,electrostatic potential map drawn and atomic charge analysis.Based on the analysis of amino acid electrostatic effects,Asp218 was considered as a candidate amino acid for subsequent mutation studies.FAcD catalyze dehalogenation of aliphatic haloacids by two part,in which Asp 110 act as the nucleophile attack the F-bearing C atom to release the halide ion and form an ester intermediate.In the next part,a water molecule hydrolyses the ester to produce products.In this paper,the natural substrate fluoroacetic acid was used as the research object,and the hydrolysis mechanism of FAcD was deeply investigated.The complete reaction mechanism of FAcD catalytic degradation process was proposed and the hydrolysis process was determined as the rate-limiting step.The paper also studied the degradation of polyfluorinated acetic acid.It was found that as for polyfluorinated substitution substrate,rate-limiting step is changed to defluoriation process.Thus subsequent research on the degradation of polyfluorinated substituted compounds still needs to focus on the defluorination process.In summary,the research in this paper clarifies the catalyze mechanism of FAcD,which provides a theoretical basis for the redesign of enzymes for the degradation of perfluorocarboxylic acids(PFCAs).