Researches On The Mechanism Of Dibenzofuran Degradation By Burkholderia Xenovorans LB400 Biphenyl Dioxygenase

Dioxins have strong carcinogenic,teratogenic and mutagenic toxicity,and are typical marine persistent organic pollutants.As a model compound,dibenzofuran is widely used in the degradation of dioxins.At present,the researches on DF biodegradation are no longer limited to the development of microbial germplasm resources,but more focused on the artificial evolution of enzymes in metabolic pathways.Burkholderia xenovorans LB400 is currently known as the natural strain with the best biphenyl degradation effect,and it is the biphenyl dioxygenase(BPDO)in this strain that plays a decisive role in the degradation process.It is an emerging bioremediation approach to use the artificially evolved and modified LB400 BPDO to degrade DF in the marine environment.So far,no mutants which can improve the degradation ability toward DF have been found in the I and II regions of LB400 BPDO.In the previous research of our laboratory,the strain LB400 was selected as the parent,and II region mutants S283M,p4-S283M and RR41-S283M were constructed.Therefore,in this study,S283M,p4-S283M and RR41-S283M were used as the research objects to determine the enzymatic kinetic parameters and metabolites of their reaction with DF,so as to identify the effect of II mutants on the catalytic performance and regiospecificity toward DF.At the same time,computer software was used to simulate the structure of the mutant protein and molecular docking was carried out to explore the mechanism of Ser283Met mutation in the II region of catalytic center affecting the catalytic performance of LB400 BPDO.The main results were given as follows:(1)Expression and purification of BphAE_LB400 and its variantsEscherichia coli C41(DE3)cells containing bphAE_LB400(wild type),bphAE_p4(mutant),bphAE_RR41(mutant),bphAE_S283M(mutant),bphAE_p4-S283M(mutant)and bphAE_RR41-S283M(mutant)genes were cultured and induced to express.The expressed product was purified by nickel column affinity chromatography,and high purity Bph A and Bph E with molecular weight of 51 KDa and 22 KDa were obtained.(2)The enzymatic kinetic parameters of BphAEs toward DF.In this study,the k_cat/K_m value of the variant BphAE_S283M and BphAE_p4-S283M toward DF were higher than those of its parents,and the k_m value of the variant BphAE_S283M,BphAE_p4-S283M toward DF were lower than those of its parents.The results showed that the mutation of Ser283Met in the II region of the catalytic center significantly improved the catalytic ability of LB400 BPDO toward DF,and the affinity between the variant BphAE_S283M,BphAE_p4-S283M and the substrate DF was enhanced.(3)The regiospecificity of BphAEs toward DF.The metabolites of BphAE_LB400 and its mutants toward DF were identified as1,2-dihydro-1,2-dihydroxy-dibenzofuran and 3,4-dihydro-3,4-dihydroxy-dibenzofuran.Compared with their respective parent BphAE_p4 and BphAE_RR41,the main metabolites of mutants BphAE_p4-S283M and BphAE_RR41-S283M toward DF did not change,they were still1,2-dihydro-1,2-dihydroxy-dibenzofuran,but the ratios of 1,2-dihydro-1,2-dihydroxy dibenzofuran and 3,4-dihydro-3,4-dihydroxy-dibenzofuran were higher than those of their respective parent.However,compared with the parent BphAE_LB400,the ratio of the two metabolites produced by the mutant BphAE_S283M toward DF was reversed,and3,4-dihydro-3,4-dihydroxy-dibenzofuran became the main metabolite.The results showed that the Ser283Met mutation located in the II region of the catalytic center changed the regiospecificity of LB400 BPDO toward DF.(4)The structure analysis of BphAEsThe binding mode of the mutant protein and DF,the cavity volume of catalytic center and the interaction between key amino acids and DF were predicted by homology modeling and molecular docking.The results showed that,compared with the parent BphAE_LB400,the orientation of DF was significantly reversed during binding the mutant protein BphAE_S283M,the oxidative attack site was changed.At the same time,the catalytic cavity volume of the reaction between BphAE_S283M toward DF is larger than that of mutants BphAEp4-S283M and BphAERR41-S283M.