Degradation Mechanism Of DDT By Burkholderia Xenovorans LB400 Biphenyl Dioxygenase And Its Mutants

Dicholodiphenyltrichloroethanes（DDT）is probably the best known and typical persistent organic pollutant in the world,which has been widely used in malaria control and agricultural deworming.They are still detected in various environmental matrices and have new input sources although their usage in agriculture has been banned in China and other countries.Numerous concerns have arisen over the past decades about the adverse environmental impacts（including harm to offshore ecosystem and human health）of DDT.DDT and its reductive dechlorination products are degraded difficultly under natural conditions,one of the reasons is the lack of effective enzyme degradation system.At present,there is no degradation enzyme system that can completely mineralize DDT.Because of the similar aromatic compounds between DDT and PCBs,biphenyl dioxygenase（BPDO）has become an important supplement to DDT degrading enzymes with the discovery of PCBs degrading bacteria.A comparison ofαsubunit protein sequences of LB400 and other strains identified four regions（designated I,II,III,and IV）in which specific sequences were consistently associated with either a broad or narrow substrate specificity.Some scholars have studied the function of amino acids in regions III and IV,but there are few studies on the function of amino acid residues in regions I and II.In order to explore the degradation characteristics and mechanism of biphenyl dioxygenase on DDT,we selected Burkholderia xenovorans LB400 biphenyl dioxygenase as parent,and the mutant Bph AE_S283M was obtained by two-step site-directed mutagenesis from Ser to Met.The degradation characteristics and mechanism of wild type and mutant were explored by comparing the catalytic performance of wild type and mutant to DDT,simulating the structure of mutant protein and molecular docking.The purpose of this study is to explore the catalytic mechanism of biphenyl dioxygenase on DDT,and to improve the substrate range of Bph AE_LB400 and its catalytic capacity for DDT.At the same time,it lays a theoretical foundation for how to expand the range of other BPDO substrates and improve the degradation rate.The main contents and results are summarized as follow:（1）Through the sequence analysis and crystal calculation of biphenyl dioxygenase,it was found that the B-factor of 280-287 fragment was higher,which indicated that the fragment had higher flexibility.Meanwhile,the conformation could be changed in the process of substrate binding,and the mutation of 283 amino acid residue could change the force between 283 amino acid itself and other amino acids in the catalytic pocket.Therefore,p ET14b[LB400-S283M-bph AE]was constructed and transformed into Escherichia coli C41.The target protein Bph AE_S283M was successfully obtained.（2）Physiology and biochemical data showed that neither Bph AE_LB400 nor the mutant Bph AE_S283M could degrade p,p’-DDT,but the mutant Bph AE_S283M could metabolize o,p’-DDT and produced two stereoisomers.Steady-state kinetics data showed that the K_m value and k_cat value of the mutants were about 4.6 mol/L and 0.1/s,and the overall k_cat/K_m value was 24.3 L/（μmol?s）,indicating that the binding ability between the mutants and DDT was enhanced.This implies that the Ser283Met mutation,which is on the flexible fragment,is the key to catalytic lumen-matching substrate conformation and is important for biphenyl dioxygenase to catalyze the double oxidation of other compounds with larger spatial conformation at the 2,3position.（3）Structural analysis revealed that the Ser283Met mutation expanded the catalytic cavity volume and altered the spatial orientation of DDT.The catalytic center of Bph AE includes amino acid residues Met231,Tyr232,Gln226,Phe227,Gln322 and His323,along with multiple negative potential sites on the protein surface.The volume of the catalytic activity of Bph AE_S283M increased,which may be conducive to the effective binding of Bph AE_S283M to DDT.The p,p’-DDT could notbe degraded by Bph AE_LB400 or Bph AE_S283M,which may be related to its symmetrical structure and spatial conformation to chlorine atoms.The chlorine atoms in the benzene ring affect the hydroxylation reaction at positions 2 and 3,which makes Bph AE_LB400 still unable to degrade o,p’-DDT.The mutation of Ser283Met makes the two adjacent carbon atoms in the reaction ring and the catalytic center of mononuclear iron atom in the middle of the reaction distance,thus resulting in the oxidation of 2,3position.Finally,the residue 267 and the fragment 247-260 are very important for determining the stereospecificity of DDT,but the mechanism of action of these residues needs to be further explored.For DDT degrading enzyme and others,site directed mutagenesis of key amino acid residues such as 283 site in region II combined with crystal structure analysis may be an effective strategy to further enhance its DDT catalytic activity.