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

The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that 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 the 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 to 6 chlorines), 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, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity. 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 a broader substrate range, the BphAES283M, BphAEp4-S283M, and BphAERR41-S283M variants were created from the parent enzymes BphAELB400, BphAEp4, and BphAERR41, respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km values of BphAES283M, BphAEp4-S283M, and BphAERR41-S283M were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3′,4,4′-tetrachlorobiphenyl (2,3′,4,4′-CB), 2,2′,6,6′-tetrachlorobiphenyl (2,2′,6,6′-CB), and 2,3′,4,4′,5-pentachlorobiphenyl (2,3′,4,4′,5-CB). We compared the catalytic reactions of BphAELB400 and its variants toward 2,2′-dichlorobiphenyl (2,2′-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges 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. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes. IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that 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 the 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 to 6 chlorines), 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, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.