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

摘要

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 BphAE_(S283M), BphAE_(p4-S283M), and BphAE_(RR41-S283M) variants were created from the parent enzymes BphAE_(LB400), BphAE_(p4), and BphAE_(RR41), respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the k _(cat)/ K_(m) values of BphAE_(S283M), BphAE_(p4-S283M), and BphAE_(RR41-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 BphAE_(LB400) 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 BphAE_(LB400) and its variants with degradation activity. BphAE_(S283M) and BphAE_(p4-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 BphAE_(LB400) 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. BphAE_(S283M) and BphAE_(p4-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.