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 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.

Mesaconase Activity of Class I Fumarase Contributes to Mesaconate Utilization by Burkholderia xenovorans

ABSTRACTPseudomonas aeruginosa,Yersinia pestis, and many other bacteria are able to utilize the C5-dicarboxylic acid itaconate (methylenesuccinate). Itaconate degradation starts with its activation to itaconyl coenzyme A (itaconyl-CoA), which is further hydrated to (S)-citramalyl-CoA, and citramalyl-CoA is finally cleaved into acetyl-CoA and pyruvate. The xenobiotic-degrading betaproteobacteriumBurkholderia xenovoranspossesses aP. aeruginosa-like itaconate degradation gene cluster and is able to grow on itaconate and its isomer mesaconate (methylfumarate). Although itaconate degradation proceeds inB. xenovoransin the same way as inP. aeruginosa, the pathway of mesaconate utilization is not known. Here, we show that mesaconate is metabolized through its hydration to (S)-citramalate. The latter compound is then metabolized to acetyl-CoA and pyruvate with the participation of two enzymes of the itaconate degradation pathway, a promiscuous itaconate-CoA transferase able to activate (S)-citramalate in addition to itaconate and (S)-citramalyl-CoA lyase. The first reaction of the pathway, the mesaconate hydratase (mesaconase) reaction, is catalyzed by a class I fumarase. As this enzyme (Bxe_A3136) has similar efficiencies (kcat/Km) for both fumarate and mesaconate hydration, we conclude thatB. xenovoransclass I fumarase is in fact a promiscuous fumarase/mesaconase. This promiscuity is physiologically relevant, as it allows the growth of this bacterium on mesaconate as a sole carbon and energy source.

Metabolism of Doublypara-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

ABSTRACTIn this work, we examined the profile of metabolites produced from the doublypara-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-inducedPandoraea pnomenusaB356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase ofBurkholderia xenovoransLB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doublypara-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doublypara-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.