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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.

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 [+ or -] 1 nmol [min.sup.-1] per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues [M.sup.237] [S.sup.238] and [S.sup.283] outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.

Research has been conducted on the biphenyl dioxygenases. The screening protocol for the catechol metabolite detection from these dioxygenases developed on the basis of catechol nonenzymatic oxidation to dark-colored metabolites is described.

The His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase was purified. The alpha or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to produce new chimeras that were purified ht-proteins. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls showed that the catalytic capacity of hybrid enzymes composed of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

A study was conducted to examine the capability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to change 3,4-dihydroxylated biphenyl metabolites. Escherichia coli M15(pREP4) and SG13009 were utilized as bacterial strains while protein characterization was carried out using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicated that the recruitment of catabolic enzymes supporting specificities supporting enzymes of the biphenyl degradation pathway may lead to the degradation of polychlorinated biphenyls.

The Comamonas testosteroni B-356 biphenyl-chlorobiphenyl dioxygenase system, including the terminal oxygenase, an iron-sulfur protein made up of an alpha subunit and a beta subunit encoded by bphA and bphE, a ferredoxin encoded by bphF, and ferredoxin reductase encoded by bphG, is analyzed. Ferredoxin and ferredoxin reductase transfer electrons from NADH to iron-sulfur protein, which in turn causes the molecular oxygen to be inserted into the aromatic substrate.

The reactivity of biphenyl dioxygenase (BPH dox) with polychlorinated biphenyls (PCBs) has been studied. BPH dox' ability to catalyze the meta-para dioxygenation of chimeric enzymes produced by exchanging the ISP (sub BPH) alpha or beta subunit of Pseudomonas sp. strain LB400 with the corresponding subunit of Comamonas testosteroni strain B-356 has also been examined. Findings show the structure of the beta subunit regulates the ability of the enzyme to catalyze the meta-para oxygenation of the substrate and its reactivity with PCBs.