Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283

Abstract

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO) catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of broader substrate range, BphAES283M, BphAEp4-S283M and BphAERR41-S283M variants were created from parent enzymes BphAELB400, BphAEp4 and BphAERR41, respectively by substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km value of BphAES283M, BphAEp4-S283M and BphAERR41-S283M significantly increased compared to their parent enzyme. Meanwhile, we determined the Steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3′,4,4′-CB, 2,2′,6,6′-CB and 2,3′,4,4′,5-CB. We compared the catalytic reaction of BphAELB400 and its variants toward 2,2′-CB, 2,5-CB and 2,6-CB. The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site, thereby its site of hydroxylation. And this was confirmed by docking experiments. We also assessed the substrates range of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6 chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. 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. Importance The segment 280-283 in BphAEs is located at the entrance of the catalytic pocket and it shows variation conformation. In previous works, the results have been suggested but never proved residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3′,4,4′-CB, 2,2′,6,6′-CB and 2,3′,4,4′,5-CB. Meanwhile, the Ser283Met substitution altered regiospecificity of BphAE toward 2,2′-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3-6 Chlorines; Table 4), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, this study provides better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

The engineered biphenyl dioxygenases enhanced the metabolism of dibenzofuran

“…Structural analysis suggested that residue 283 located at the entrance of the catalytic cavity, was a key determinant of the catalytic reaction. Our recent reports identified that the single substitution Ser283Met of BphAE LB400 enhanced the catalytic competence toward 2,3 ′ ,4,4 ′ -tetrachlorobiphenyl, 2,2 ′ ,6,6 ′ -tetrachlorobiphenyl and 2,3 ′ ,4,4 ′ ,5-tetrachlorobiphenyl (Li et al, 2020). Although the evolved BphAE S283M , BphAE p4-S283M , BphAE RR41-S283M exhibited an enhanced ability to transform polychlorobiphenyl, their catalytic features toward dibenzofuran had never been evaluated.…”

“…Escherichia coli C41(DE3) (Statagene, La Jolla, CA) was used in this study (Miroux, 1996). In previous work, we evolved BphAE LB400 , BphAE p4 and BphAE RR41 , obtaining BphAE S283M , BphAE p4-S283M and BphAE RR41-S283M (Li et al, 2020).…”

“…In our previous work, the new mutants (BphAE S283M , BphAE p4-S283M and BphAE RR41-S283M )， were obtained by the single substitution Ser283Met in BphAE LB400 , BphAE p4 and BphAE RR41 (see Table 1). The new mutants exhibited the superior ability to metabolize biphenyl and PCBs (Li et al, 2020).…”

Augmentation of an Engineered Bacterial Strain Potentially Improves the Cleanup of PCB Water Pollution

“…We did not focus only on the ecology of aerobic PCB-degrading bacterial strains but also considered the substrate specificity of biphenyl dioxygenases (BphA), which catabolize PCB congeners and are important factors in the trial of this bioaugmentation model. There have been many reports on the substrate specificity of BphA for PCBs, and there have been multiple attempts to genetically modify the enzyme ( 24 – 28 ). The substrate specificity of BphA for PCB congeners can be roughly divided into two types of biphenyl dioxygenase activities; one is a biphenyl-3,4-dioxygenase activity from a B. xenovorans LB400 strain, and the other is a biphenyl-2,3-dioxygenase activity from another aerobic PCB-degrading bacterial strain, including the LB400 strain.…”

“…BphA determines the range of PCB congeners oxidized and plays an essential role in substrate recognition and binding ( 29 ). Genetically modified BphA containing a motif of the BphA gene of the LB400 strain has been investigated in detail ( 26 – 28 ). We, therefore, constructed a recombinant PCB-degrading bacterium that produced the BphA of the LB400 strain using E. coli as a host strain and confirmed that its substrate specificity was biphenyl-3,4-dioxygenase ( Fig.…”