Resolving the profile of metabolites generated during oxidation of dibenzofuran and chlorodibenzofurans by the biphenyl catabolic pathway enzymes

Although the metabolism of dibenzofuran by the biphenyl catabolic enzymes had been inferred in previous reports, the metabolic pattern has never been determined unambiguously. In this work, we describe the evolved biphenyl dioxygenase (BPDO) RR41 that exhibits a higher turnover rate of metabolism toward dibenzofuran and chlorodibenzofurans than the parental Burkholderia xenovorans LB400 BPDO. We used RR41 BPDO to identify unambiguously the metabolites produced from the oxygenation of dibenzofuran by LB400 BPDO, and we evaluated their further metabolism by the biphenyl catabolic pathway enzymes of strain LB400. RR41 BPDO was obtained by saturation mutagenesis of targeted amino acid residues. (IFNIL409)-F-335-N-336-I-338-L-341 of LB400 BphA were replaced by A(335)M(336)Q(338)V(341)F(409) in RR41 BphA. Data confirm the critical role played by these amino acid residues for substrate specificity and regiospecificity.