Engineering The Biphenyl Dioxygenase Of Burkholderia Xenovorans LB400

Polychlorinated biphenyls?PCBs?are persistent pollutants and typical marine pollutants.They have been evaluated to affect human health and ecosystems,resulting from their hydrophobic properties and accumulation in the coastal zone.Burkholderia xenovorans LB400,which lives in a wide range of environments including soil and marine,is one of the most effective strains of natural origin.Engineering B.xenovorans LB400 is an effective strategy to remediate marine pollution of PCBs.Polychlorinated biphenyls are commonly metabolized via the bacterial biphenyl degradation pathway and the substrate range of the biphenyl dioxygenase?BPDO?initiating the catabolism determines the capacity of BPDO against various congeners.The substrate range and regiospecificity of BPDO are principally determined by residues located on the C-terminal portion of the dioxygenase?subunit which was designated as regions I,II,III,and IV.To date,the improvement in biphenyl dioxygenase has been investigated extensively in regions III and IV,but little is known about evolving BPDO in regions I and II.Consequently,we mutated Ser283 of BphAE_LB400,BphAE_p4 and BphAE_RR41 to Met as in BphAE_B356 to generate BphAE_S283M,BphAE_p4-S283M and BphAE_RR41-S283Mrespectively.We compared some of the catalytic properties of BphAE_LB400,BphAE_p4,BphAE_RR41,BphAE_S283M,BphAE_p4-S283M and BphAE_RR41-S283M towards biphenyl and selected PCB congeners and we also modeled the structures of BphAE_S283M,BphAE_p4-S283M and BphAE_RR41-S283Mand docked substrates in order to investigate structural features of BphAEs responsible for the differential catalytic properties of these enzymes.The main contents and results are summarized as follow:1. Two-step site-directed mutagenesis protocol was utilized to construct three mutants with the single substitution Ser283Met in BphAE_LB400,BphAE_p4and BphAE_RR41.BphAEs were expressed in E.coli C41?DE3?as His-tagged protein and purified by affinity chromatography.The level of expression of purified enzymes was assessed by SDS-PAGE.Two single bands were observed corresponding to the??Bph A,Mr=51,000?and??Bph E,Mr=22,000?subunits.2. The steady-state kinetic parameters of BphAE_LB400 and its variants toward PCBs were calculated from the oxygen consumption using an oxygraph. The results of steady-state kinetic parameters showed that for biphenyl,the k_cat/K_m value of BphAE_S283M,BphAE_p4-S283M and BphAE_RR41-S283M significantly increased compared to their parent enzyme.To our best knowledge,the kinetics of the reaction of most engineered described BphAEs toward biphenyl was lower than their parental enzymes.However,in this work,the k_cat/K_mvalue of mutants BphAE_S283M,BphAE_p4-S283M and BphAE_RR41-S283M towards biphenyl was higher compared to their respective parent.Meanwhile,this is the first report investigating the steady-state kinetics of BphAEs toward highly chlorinated biphenyls.The results indicated that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward2,3',4,4'-CB,2,2',6,6'-CB and 2,3',4,4',5-CB.Thus,for BphAEs displaying structural feature similar to that of BphAE_LB400,the Ser283Met mutation of BphAE_LB400variants may be a strategy to further enhance their catalytic activity toward biphenyl and highly chlorinated biphenyls.3. The regiospecificity of BPDO influences the formed products that influence the catalytic degradation of the substrates by the subsequent enzymes of the pathway. We compared the catalytic reaction of BphAE_LB400 and its variants toward 2,2'-CB,2,5-CB and 2,6-CB.The metabolites were identified by GC-TOF-MS analysis.The fact that the ratio of 2,3-dihydro-2,3-dihydroxy-2'-chlorobiphenyl to3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl was higher in all the Ser283Met variants compared to their parental enzyme indicates that the regiospecificity toward2,2'-dichlorobiphenyl was influenced by the Ser283Met substitution.In the case of2,6-CB,the metabolite ratio was inversed compared to BphAE_p4and BphAE_RR41 in which larger amounts of 3,4-dihydroxylated metabolites were produced in the variant enzymes.Additionally,the 3D structures of the wild-type and mutant enzymes were modeled and molecular docking was performed to explain the new properties of the mutant enzymes.The biochemical data indicated that the Ser283Met substitution alters the orientation of the substrate inside the catalytic cavity,thereby its site of hydroxylation.And this was confirmed by docking experiments.4. We assessed the capacity of recombinant E. coli DH11S cells with Bp AE_LB400and its variants to degrade PCB congeners.BphAE_S283M metabolized a broader range of the 11 congeners.BphAE_S283M exhibited an enhanced ability to metabolize the 10 congeners compared to BphAE_p4 and Bp AE_RR41,indicating a meaningful contribution of Met283 of BphAE_S283M to the selectivity of some PCB substrates.Notably,BphAE_S283M and BphAE_p4-S283Mwere clearly improved in oxidizing some of the 3-6 chlorinated biphenyls,which are generally very poorly oxidized by most dioxygenases.Thus,S283M and p4-S283M have a potential application in remediation of PCBs pollution in the coastal zone of China.Collectively,the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO.It will certainly be meaningful elements for understanding the effect of the residue corresponding to 283 in other Rieske oxygenase enzymes.This study provides better insights into the mechanisms by which to evolve BPDO to change and/or expand its substrate range and its regiospecificity.Additionally,this study provides a theoretical basis and technical support for remediating marine pollution of PCBs.