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Research has been conducted on the cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356. The substitution of each triad residue has been investigated in BphB.

Sequence analysis of Comamonas testosteroni strain B-356 bphB shows that 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (B2,3D) is as active as other bacterial strains in its ability to degrade polychlorinated biphenyl (PCB) congeners. B2,3D requires NAD+, an optimal pH of 9.5, and a native M(sub r) of 123, 000. These features are similar to those of the related molecule cis-toluene dihydrodiol dehydrogenase. However, unlike cis-toluene dihydrodiol dehydrogenase, pattern B356 B2,3D is unable to transform cis-1,2-dihydroxycyclohexa-3,5-diene.

Results indicate that between the two dihydroxybiphenyls, 3,3'-dihydroxybiphenyl is the preferred substrate for the biphenyl catabolic enzymes of Comamonas testosteroni B-356. Data show that in the metabolic pathway of the dihydroxybiphenyls, the major step is a direct dehydroxylation of one of the ortho-substituted carbons yielding 2,3,2'-trihydroxybiphenyl.

Despite the wide utility of hydroxylamines in organic synthesis, relatively few are commercially available, and there is a need for direct, efficient, and selective methods for their synthesis. Herein, we report two complementary methods to accomplish direct oxidation of secondary amines using UHP as an oxidant. The first method uses 2,2,2-trifluoroethanol (TFE) and a large excess of amine. Isolation of hydroxylamine products is enabled by selective salt formation, and recovery of excess amine is demonstrated. The second method uses hexafluoroacetone as an additive and is highlighted by the 1:1 stoichiometry between the oxidant and amine.

While attempting to synthesize biaryl ethers we discovered the inadvertent formation of a methylsulfoxylmethyl ether byproduct. Formation of this unexpected byproduct presented an opportunity to streamline the synthesis of methylsulfoxylmethyl ethers. Mechanistic studies suggest a radical pathway with dimsyl potassium as a reducing agent.

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7 × 10⁵ CFU g⁻¹) was greater than the number remaining in the surrounding sand (4.5 × 10⁴ CFU g⁻¹). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4'- and 2,3',4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria-Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE(RR41), a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE(LB400), metabolized a broader range of PCBs than BphAE(LB400). Hence, BphAE(RR41) was able to metabolize 2,6,2',6'-, 3,4,3',5'- and 2,4,3',4'-tetrachlorobiphenyl that BphAE(LB400) is unable to metabolize. BphAE(RR41) was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE(LB400) to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the alpha subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity., (Copyright 2008 S. Karger AG, Basel.)

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (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(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(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(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(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 +/- 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.

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.

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.

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.

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.

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. 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 create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(LB400)beta(P6), and ht-alpha(B-356)beta(P6). ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E. coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1, 2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2, 3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl to 2, 3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3, 4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl (3,4-DH-2,2',5,5'-TCB), respectively. Our data also suggested that purified 1, 2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3, 4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3, 4-DH-2,2',5,5'-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.

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 (alpha and beta) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). 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 ISPBPH alpha or beta 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 alpha subunit of ISPBPH of strain LB400 and the beta subunit of ISPBPH of strain B-356 (the alphaLB400 betaB-356 chimera) and the alphaB-356betaLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH beta subunit influences BPH dox's reactivity pattern toward chlorobiphenyls. Thus, if the alpha subunit were the sole determinant of the enzyme reactivity pattern, the alphaB-356betaLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alphaLB400 betaB-356 chimera showed features of both B-356 and LB400 ISPBPH 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.

In addition to 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O), biphenyl-grown cells of Comamonas testosteroni B-356 were shown to produce a catechol 2,3-dioxygenase (C2,3O). B1,2O showed strong sequence homology with B1,2Os found in other biphenyl catabolic pathways, while partial sequence analysis of the C2,3O of B-356 suggested a relationship with xylEII-encoded C2,3O. The coexistence of two meta-cleavage dioxygenases in this strain prompted a comparison between the catalytic properties of the two enzymes. C2,3O has a much broader substrate specificity than native or His-tagged B1,2O: both enzymes were inhibited by chlorocatechols, but B1,2O was more sensitive than C2,3O. The results are discussed in terms of the physiological implications of interaction between metabolites from the lower biphenyl-chlorobiphenyl pathway and enzymes of the upper pathway.

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.

Pseudomonas testosteroni B-356 was able to degrade approximately 50% of the Aroclor 1242 mixture in shaken culture. The aims of the present study were to evaluate the capabilities of this bacterial strain to degrade PCBs in soil microcosms and to identify some of the factors likely to favor the degradative performance of the implanted bacteria. The presence of biphenyl as cosubstrate was the most important factor affecting PCB degradation in soil. However, because biphenyl was rapidly depleted in soil microcosms, repeated addition of small amounts of biphenyl to maintain a constant level of the cosubstrate allowed the achievement of a higher degree of degradation of the tetrachlorinated components of Aroclor 1242 than was achieved with a single addition at the time of inoculation. Degradation of di- and tri-chlorinated PCB congeners was less affected by repeated addition of biphenyl because these congeners were degraded very fast and complete degradation was achieved before biphenyl was depleted in the soil. Biodegradation was also related to bioavailability of the substrate. We observed that the proportion of each congener degraded was higher in the microcosms receiving both the producer of the surface-active agent, Alcaligenes faecalis B-556, and strain B-356. Under the best conditions (presence of a constant level of biphenyl and of strain B-556) P. testosteroni B-356 was able to degrade approximately 30% of the Aroclor 1242 added to soil microcosms; some other factors reducing the PCB degradation capabilities of the implanted bacteria are also discussed.