Characterization of biphenyl dioxygenase of Pandoraea pnomenusa B-356 as a potent polychlorinated biphenyl-degrading enzyme

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenylls (PCBs). In three different assays, BPDOB356 from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDOLB400 from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro similar to 2.,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro similar to 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro similar to 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetractilorobiphenyl, BPDOB356 transformed each congener at a higher rate than BPDOLB400. The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDOB356 activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDOLB400 had a greater apparent specificity for biphenyl than BPDOB356 (k(cat)/K-m =2.4 x 10(6) +/- 0.7 x 10(6) M-1 s(-1) versus k(cat)/K-m = 0.21 x 10(6) +/- 0.04 x 10(6) M-1 s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO,B,, containing four active site residues of BPDOB356 transformed para-substituted congeners better than BPDOLB400. Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O-2 consumption was approximately proportional to congener depletion. At 2.4-angstrom resolution, the crystal structure of the BPDOB356-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.