Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.     

Biphenyl dioxygenase from an arctic isolate is not cold adapted

Biphenyl dioxygenase from the psychrotolerant bacterium Pseudomonas sp. strain Cam-1 (BPDO(Cam-1)) was purified and found to have an apparent k(cat) for biphenyl of 1.1 +/- 0.1 s(-1) (mean +/- standard deviation) at 4 degrees C. In contrast, BPDO(LB400) from the mesophile Burkholderia xenovorans LB400 had no detectable activity at this temperature. At 57 degrees C, the half-life of the BPDO(Cam-1) oxygenase was less than half that of the BPDO(LB400) oxygenase. Nevertheless, BPDO(Cam-1) appears to be a typical Pseudomonas pseudoalcaligenes KF707-type dioxygenase.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Family shuffling of soil DNA to change the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase

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 +/- 1 nmol min(-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 M237 S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.     

Substrate selectivity pattern of Comamonas testosteroni strain B-356 towards dichlorobiphenyls

In this work, we have investigated the substrate selectivity pattern of strain B-356 resting cell suspensions and cell lysates towards selected chlorobiphenyl congeners. The strain showed a preference for the double meta-substituted congener 3,3′-dichlorobiphenyl over the double ortho-substituted congener 2,2′-dichlorobiphenyl and the double para-substituted congener 4,4′-dichlorobiphenyl. The results are discussed with reference to the substrate selectivity pattern reported for Pseudomonas sp. strain LB400.     

Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP_BPH), a ferredoxin (FER_BPH), and a ferredoxin reductase (RED_BPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISP_BPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP_BPH of strain LB400 and the β subunit of ISP_BPH of strain B-356 (the α_LB400β_B-356 chimera) and the α_B-356β_LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP_BPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α_B-356β_LB400 chimera should have behaved like B-356 ISP_BPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP_BPH. On the other hand, the α_LB400β_B-356 chimera showed features of both B-356 and LB400 ISP_BPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.     

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 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 of Burkholderia xenovorans LB400 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 doubly para-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 doubly para-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.     

Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.     

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE_LB400) are largely unknown. BphAE_p4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE_LB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE_LB400 and BphAE_p4 and examine the biochemical properties of two BphAE_LB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.     

Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni B-356.

The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 alpha subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO alpha subunit with that of LB400 alpha subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.     

Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase

In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp. strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6. Unlike all parents, these variants exhibited high activity toward 2,2'-, 3,3'-, and 4,4'-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (335TFNNIRI341) of LB400 BphA by the corresponding segment (333GINTIRT339) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.     

Metabolism of dibenzofuran and dibenzo-p-dioxin by the biphenyl dioxygenase of Burkholderia xenovorans LB400 and Comamonas testosteroni B-356

We examined the metabolism of dibenzofuran (DF) and dibenzo-p-dioxin (DD) by the biphenyl dioxygenase (BPDO) of Comamonas testosteroni B-356 and compared it with that of Burkholderia xenovorans LB400. Data showed that both enzymes oxygenated DF at a low rate, but Escherichia coli cells expressing LB400 BPDO degraded DF at higher rate (30 nmol in 18 h) compared with cells expressing B-356 BPDO (2 nmol in 18 h). Furthermore, both BPDOs produced dihydro-dihydroxy-dibenzofuran as a major metabolite, which resulted from the lateral oxygenation of DF. 2,2,3-Trihydroxybiphenyl (resulting from angular oxygenation of DF) was a minor metabolite produced by both enzymes. Deuterated DF was used to demonstrate the production of 2,2,3-dihydroxybiphenyl through angular oxygenation of DF. When tested for their ability to oxygenate DD, both enzymes produced as sole metabolite, 2,2,3-trihydroxybiphenyl ether at about the same rate, indicating similar catalytic properties toward this substrate. Altogether, although LB400 and B-356 BPDOs oxygenate a different range of chlorobiphenyls, their metabolite profiles toward DF and DD are similar. This suggests that co-planarity influences the regiospecificity of BPDO toward DF and DD to a higher extent than the presence of an ortho substituent on the molecule.     

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates.

Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively). A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase.     

Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.     

Remarkable ability of Pandoraea pnomenusa B356 biphenyl dioxygenase to metabolize simple flavonoids

Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to degrade chlorobiphenyls in soil. However, the bases for this interaction are largely unknown. In this work, we found that BphAE(B356), the biphenyl/chlorobiphenyl dioxygenase from Pandoraea pnomenusa B356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAE(LB400) from Burkholderia xenovorans LB400. Unlike those of BphAE(LB400), the kinetic parameters of BphAE(B356) toward these flavonoids were in the same range as for biphenyl. In addition, remarkably, the biphenyl catabolic pathway of strain B356 was strongly induced by isoflavone, whereas none of the three flavonoids induced the catabolic pathway of strain LB400. Docking experiments that replaced biphenyl in the biphenyl-bound form of the enzymes with flavone, isoflavone, or flavanone showed that the superior ability of BphAE(B356) over BphAE(LB400) is principally attributable to the replacement of Phe336 of BphAE(LB400) by Ile334 and of Thr335 of BphAE(LB400) by Gly333 of BphAE(B356). However, biochemical and structural comparison of BphAE(B356) with BphAE(p4), a mutant of BphAE(LB400) which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAE(LB400), provided evidence that other residues or structural features of BphAE(B356) whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAE(B356). Together, these data provide supporting evidence that the biphenyl catabolic pathways have evolved divergently among proteobacteria, where some of them may serve ecological functions related to the metabolism of plant secondary metabolites in soil.     

Differential enantioselective transformation of atropisomeric polychlorinated biphenyls by multiple bacterial strains with different inducing compounds

Five polychlorinated biphenyl (PCB)-degrading bacteria were tested for the ability to differentiate between the enantiomers of four atropisomeric PCB congeners (2,2',3,6-tetra-CB; 2,2',3,3',6-penta-CB; 2,2',3,4',6-penta-CB; and 2,2',3,5',6-penta-CB) after growth in the presence of tryptone-soytone, biphenyl, carvone, or cymene. Enantioselectivity was shown to vary with respect to strain, congener, and cosubstrate.     

Congener Selectivity During Polychlorinated Biphenyls Degradation by Enterobacter sp. LY402

The relationship between the selectivity of a particular polychlorinated biphenyls (PCBs) congener and its biodegradability under the same concentration, especially by Enterobacter sp. LY402, is less well studied. To measure congener selectivity of Enterobacter sp. LY402, several influencing factors were studied. The results showed LY402 effectively degraded coplanar 3,4,3',4'-chlorobiphenyl (CB) at a concentration of 0.05 μM, but not 0.5 μM. The degradation rates of 2,4,5,2',3'-CB and 2,4,5,2',4',5'-CB were increased significantly when the sample constituents were changed from 12 to 5 congeners or to one congener. This indicated that bioremediation of individual congener was affected by other congeners present in the mixture. Moreover, for PCBs containing one chlorine on each phenyl ring, the reactivity preference of LY402 was 2,2'-CB ≥ 3,3'-CB ? 4,4'-CB. For two ortho chlorines congeners of PCBs, 2,2'-CB was degraded faster than 2,6-CB. Although 2,6-CB and 4,4'-CB were poorly degraded, the addition of one (i.e., 2,4,4'-CB and 2,6,3'-CB) or two more chlorines (i.e., 2,4,2',4'-CB) on the phenyl ring significantly increased their biodegradability. In addition, comparing the two congeners of ortho-meta-chlorinated biphenyl, 2,3,2',3'-CB with neighbor meta chlorines was degraded slower than 2,5,2',5'-CB with interval meta chlorines. All these indicated that the transformation rates of PCBs were not consistent with the number of chlorines, and PCBs containing the same numbers of chlorines but at different positions also resulted in different conversions. In principle, the extents of effect caused by the position of chlorine substituents on the degradation of PCBs by LY402 were ortho- > meta- > para-CB. In conclusion, the congener selectivity of LY402 was determined by many factors, including the composition of the congeners, their concentrations in the mixture and location and number of chlorine substituents on the phenyl rings.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation.

The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria. The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners. Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners. BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity. The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV. Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707. Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners. However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative. These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase.     

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.