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 biphenyls (PCBs). In three different assays, [BPDO.sub.B356] from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than [BPDO.sub.LB400] from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro ~2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro ~2,2'-dichloro > 2,6-dichioro > 2~2',3,3'-tetrachloro ~2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, [BPDO.sub.B356] transformed each congener at a higher rate than [BPDO.sub.LB400]. The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized [BPDO.sub.B356] activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. [BPDO.sub.LB400] had a greater apparent specificity for biphenyl than [BPDO.sub.B356] ([k.sub.cat]/[K.sub.m] = 2.4 x [10.sup.6] [+ or -] 0.7 x [10.sup.6] [M.sup.-1] [s.sup.-1] versus [k.sub.cat]/[K.sub.m] = 0.21 x [10.sup.6] [+ or -] 0.04 x [10.sup.6] [M.sup.-1] [s.sup.-1]). However, the latter transformed biphenyl at a higher maximal rate ([k.sub.cat] = 4.1 [+ or -] 0.2 [s.sup.-1] versus [k.sub.cat] = 0.4 [+ or -] 0.1 [s.sup.-1]). A variant of [BPDO.sub.LB400] containing four active site residues of [BPDO.sub.B356] transformed para-substituted congeners better than [BPDO.sub.LB400]. 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.sub.2] consumption was approximately proportional to congener depletion. At 2.4-[Angstrom] resolution, the crystal structure of the [BPDO.sub.B356]-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.