INTRODUCTION

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Aryl hydroxylating dioxygenases catalyze the first enzymatic step for most bacterial catabolic pathways involved in the degradation of aromatic compounds (28). They catalyze a dihydroxylation reaction onto vicinal carbons of the aromatic ring. These enzymes can catalyze the hydroxylation of several substrate analogs, which makes them potentially useful for the development of biocatalytic processes to destroy persistent pollutants such as polychlorinated biphenyls (PCBs). Biphenyl dioxygenase (BPH dox) can catalyze the hydroxylation of several PCB congeners, but to extend its capacity to hydroxylate more persistent congeners, new engineered enzymes will need to be developed.

BPH dox has been purified from Comamonas testosteroni B-356 (18, 19) and from Pseudomonas sp. strain LB400 (8, 13, 14). It comprises three components (8, 13, 14, 18, 19): the terminal oxygenase, an iron-sulfur protein (ISP_BPH) made up of an  (M_r = 51,000) and a  (M_r = 22,000) subunit; a ferredoxin (FER_BPH; M_r = 12,000); and a ferredoxin reductase (RED_BPH; M_r = 43,000). The encoding genes for both strain B-356 and strain LB400 are bphA (ISP_BPH  subunit), bphE (ISP_BPH  subunit), bphF (FER_BPH), and bphG (RED_BPH) (11, 35). BPH dox hydroxylates vicinal ortho-meta carbons of one of the BPH rings to generate 2,3-dihydro-2,3-dihydroxybiphenyl. FER_BPH and RED_BPH are involved in electron transfer from NADH to ISP_BPH, which is directly involved in the catalytic oxygenation of the molecule (19). The enzyme is also found in the genus Rhodococcus. The four genes that code for Rhodococcus globerulus P6, Rhodococcus strain RHA1, and Rhodococcus sp. strain M5 BPH dox have been sequenced (2, 27, 37). They are designated bphA1 ( subunit), bphA2 ( subunit), bphA3 (FER_BPH), and bphA4 (RED_BPH) in strain P6 (2).

In previous work, rhodococcal BPH dox was poorly expressed in Escherichia coli (27, 29). However, the genes encoding rhodococcal BPH dox components were expressed in recombinant Pseudomonas (29) and Rhodococcus (27). The substrate selectivity patterns of Pseudomonas putida KT2442 carrying the genes coding for strain P6 BPH dox were analyzed by testing the catalytic capacity of resting cell suspension on 3,4'-dichlorobiphenyl and 2,2'-dichlorobiphenyl. Data suggested that the enzyme metabolizes preferentially the meta-substituted ring over the para-substituted and poorly transformed the double-ortho-substituted congener 2,2'-dichlorobiphenyl (29). Furthermore, P6 BPH dox was unable to catalyze the hydroxylation of 2,2',5,5'-tetrachlorobiphenyl used to determine the capacity of the enzyme to catalyze the meta-para hydroxylation of the biphenyl molecule (29). Coincidentally, these features are similar to those reported for strain B-356 BPH dox (17). Unlike these two strains, strain LB400 BPH dox shows a preference for 2,2'-dichlorobiphenyl, poorly transforms 3,3'-dichlorobiphenyl, and is able to catalyze the meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl (15, 17, 22, 30).

Although Pseudomonas pseudoalcaligenes KF707 and LB400 BPH doxes components show a high level of identity, their substrate selectivity patterns toward chlorobiphenyls are quite distinct. Only few amino acid residues located at the N-terminal portion of the terminal oxygenase  subunit were found to determine the substrate specificity of these enzymes (22, 30). Based on sequence comparison between enzymes of several strains, Mondello et al. (30) have identified four regions of the terminal oxygenase  subunit in which specific sequences were consistently associated with either broad (LB400-type) or narrow (KF707-type) PCB substrate specificity. Based on published data of DNA sequence and substrate specificity (2, 29), like B-356 ISP_BPH, P6 ISP_BPH correlates with the KF707-type strain.

Recently, His-tagged purified chimeras (ht-chimeras) were obtained by exchanging the  and  subunits of LB400 and B-356 BPH dox terminal oxygenases (17). The amino acid residues of the  subunit which were found to determine the substrate selectivity pattern of LB400 and KF707 terminal oxygenase (22, 30) were not found to influence the substrate selectivity pattern of the engineered chimeras (17). Furthermore, the substrate selectivity pattern of ISP_BPH chimeras comprised of B-356  subunit with LB400  subunit (_B-356LB400) was very similar to that of LB400 BPH dox, which suggests an involvement of the  subunit on the reactivity pattern of the terminal oxygenase toward PCBs. To extend this study, in this work we purified recombinant P6 ht-ISP_BPH expressed from E. coli and Pseudomonas and compared its substrate selectivity pattern toward chlorobiphenyls with that of engineered purified ht-ISP_BPH chimeras obtained by exchanging P6  or  subunit with corresponding peptide of LB400 or B-356 ISP_BPH.

	MATERIALS AND METHODS

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Bacterial strains, culture media, and general protocols. The bacterial strains used in this study were E. coli M15[pREP4] and SG13009[pREP4] (both from Qiagen Inc., Chatsworth, Calif.), E. coli DH11S (25), P. putida KT2440 (4), C. testosteroni B-356 (1), Pseudomonas sp. strain LB400 (6) (also referred as Burkholderia sp. strain LB400 or Pseudomonas cepacia LB400 [22]), and R. globerulus P6 (2, 3, 12). The media used were Luria-Bertani (LB) broth or solidified with agar (33). Plasmid DNA from E. coli, restriction endonuclease reactions, ligations, agarose gel electrophoresis, and transformation of E. coli cells were done according to protocols described by Sambrook et al. (33). The transformation of P. putida KT2440 was done according to the protocol described by Sambrook et al. (33) for the transformation of E. coli except that Ca²⁺ was replaced by Rb²⁺ and the cells were incubated for 2 h at 28°C (instead of 45 min at 37°C) before plating. The transformation rates were approximately 10² transformants per µg of DNA added to the ligation reaction medium. PCR was performed with Pwo DNA polymerase according to the method recommended by Boehringer Mannheim.

Plasmids. Several vector and plasmids were used in this study. Plasmid pQE31, designed to create His-tagged fused proteins, and pQE51, which is identical to pQE31 except for the lack of the His₆-tagged fused gene, were both from Qiagen. Plasmid pYH31 (17, 20) is also designed to create His-tagged fused proteins but is compatible with ColE1-based plasmids. Plasmid pEP31, obtained during this work (Fig. 1A), is a shuttle vector designed to produce fusion ht-protein in Pseudomonas and in E. coli. It confers resistance to both tetracycline and ampicillin. For construction of pEP31, pUCP26 (10, 34), which was graciously provided by H. P. Schweizer (Department of Microbiology, Colorado State University, Fort Collins), was treated with PvuI. The 3.2-kb fragment carrying the rep region (ori [origin of replication] of Pseudomonas aeruginosa PAO, which allows the plasmid to replicate in Pseudomonas) and tetracycline resistance (pALTER-1) was made blunt ended. Similarly, the 2.5-kb PvuII/NdeI fragment of pQE31 carrying the ori for replication, amplicillin resistance, and the promoter-operator region plus the His-tagged fusion gene was made blunt ended and then ligated with the 3.2-kb DNA fragment from pUCP26. Likewise, for construction of pEP51, the 2.5-kb PvuII/NdeI fragment of pQE51 was ligated to the 3.2-kb PvuI fragment of pUCP26. For construction of the 7.8-kb plasmid pQE51[LB400-bphFGBC], a 3.5-kb SmaI/NdeI fragment from pQE51 was ligated to a 4.3-kb BbrPI/NdeI fragment which carries LB400 bphFGBC from pAH17 (graciously provided by V. De Lorenzo, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Crentíficas Madrid, Spain). Similarly, for construction of the 8.3-kb plasmid pYH31[LB400-bphFGBC], the 4.3-kb BbrPI/NdeI fragment from pAH17 was ligated blunt end to the 4-kb plasmid pYH31 digested with KpnI at its unique KpnI site.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   (A) Linear map of the E. coli-Pseudomonas shuttle vector pEP31 designed to produce ht-proteins. Only selected restriction sites are shown. The grey and white areas derive from pUCP26 and pQE31, respectively. rep, origin of replication in Pseudomonas; tet, tetracycline resistance; bla, ampicillin resistance; ori, ColE1 origin of replication; pro/op, pQE31 promoter-operator; 6xHis, the six-histidine-tagged fusion gene. The map of pEP51 is identical to that of pEP31 except for the absence of the six-histidine-tagged fusion gene. (B) Constructs used to produce P6 ht-ISPBPH and chimeras derived from it. All constructs except pEP31[P6-bphA1/p-o/bphA2] were made in pQE31, and the cloned DNA fragment was transferred to pEP31 when needed. Details of the strategies used to construct these plasmids are given in Materials and Methods. Only the restriction sites important for the cloning strategies are shown. KpnI/K and XhoI/K indicate sites that were made blunt ended with the Klenow fragment of DNA polymerase I, resulting in their loss in the final construct.

Plasmid constructions used to express P6 ht-ISP_BPH and ht-ISP_BPH chimeras. In this work we have produced purified preparations of P6 ht-ISP_BPH (ht-_P6P6) and of all four hybrid combinations between the P6 terminal oxygenase  and  subunits and the corresponding subunits of LB400 and B-356 terminal oxygenases, thus producing ht-_P6LB400, ht-_LB400P6, ht-_P6B-356, and ht-_B-356P6.

Expression and purification of P6 ISP_BPH and its chimeras in E. coli and P. putida KT2440. The ht-enzymes were expressed in E. coli cells according to protocols described previously (18, 19). When the proteins were expressed from pEP31 in E. coli cells, the antibiotic concentrations used were 200 µg of ampicillin per ml and 10 µg of tetracycline per ml (tet 10).

Protein characterization. Sodium dodecyl sulfate (SDS)-polyacrylamide gels were developed according to method of Laemmli (24). Proteins were stained with Coomassie brillant blue (33). Protein concentrations were estimated by the method of Lowry (26), using bovine serum albumin as a standard. The concentrations of all ht-ISP_BPH preparations were also determined spectrophotometrically, using the ₄₅₅ value of 8,300 M¹ cm¹ established for B-356 ht-ISP_BPH (18). The preparations of B-356 ht-FER_BPH and B-356 ht-RED_RED used in this work were also quantified spectrophotometrically as previously described (19). The M_r of the native proteins was determined by high-pressure liquid chromatography (HPLC) as described previously (18).

Monitoring of enzyme activities and identification of metabolites. Enzyme assays for BPH dox were performed as described previously (17). The reaction was initiated by adding 50 nmol of biphenyl or 25 nmol of one of the following chlorobiphenyls: 2,2'-, 3,3'-, or 2,5-dichlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl (all from ULTRAScientific, Kingstown, R.I.) (added in 2 µl of acetone). Based on previous data showing that the origin of FER_BPH did not influence the BPH dox substrate reactivity pattern (17), the reconstituted BPH doxes comprised either P6 ht-ISP_BPH or one of the ht-ISP_BPH chimeras described above (_P6LB400, _LB400P6, _P6B-356, or _B-356P6) plus B-356 ht-FER_BPH and B-356 ht-RED_BPH. The catalytic oxygenation was evaluated by monitoring substrate depletion by HPLC analysis 5 or 10 min after initiation of the reaction, as described previously (17). When 2,2',5,5'-tetrachlorobiphenyl was the substrate, the catalytic oxygenation was evaluated by monitoring the metabolite production by HPLC using the conditions described previously (5).

	RESULTS

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Stability of pEP31 and pEP51 in E. coli and Pseudomonas. Plasmids pEP31 and pEP51 were maintained for over 50 generations in E. coli DH11S grown on tet 10 as well as in P. putida KT2440 grown on tet 20. As shown by data in Fig. 2, the cloned DNA fragment carrying P6 bphA1/p-o/bphA2 was stably maintained for over 50 generations both in E. coli and in Pseudomonas since both subunits of P6-ht-ISP_BPH were produced in those recombinants. Same results were obtained with P6-bphA1/LB400-bphE or P6-bphA1/B-356-bphE.

View larger version (96K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE of P6 ht-ISPBPH or P6 ISPBPH produced in E. coli or P. putida KT2440 carrying pEP31[P6-bphA1/p-o/bphA2] or pEP51[P6-bphA1/p-o/bphA2]. Lane 1, crude extract (1.9 µg of protein) of E. coli DH11S carrying pYH31[LB400-bphFGBC] plus pEP31[P6-bphA1/p-o/bphA2], where protein induction was achieved as described in Materials and Methods except that the inoculum was a culture grown for 50 generations in LB broth (tet 10); lane 2, purified preparation (1.1 µg of protein) of ht-P6P6 obtained from the extract in lane 1; lane 3, Mr marker; lane 4, crude extract (2.4 µg of protein) of P. putida KT2440 carrying pEP31[P6-bphA1/p-o/bphA2] where the inoculum was grown for 50 generations in LB broth (tet 20); lane 5, purified preparation (0.1 µg of protein) of ht-P6P6 obtained from the extract shown in lane 4; lane 6, crude extract (8 µg of protein) of P. putida KT2440 carrying pEP51[P6-bphA1/p-o/bphA2] grown for 50 generations as in lane 4; lane 7, crude extract (1 µg of protein) of E. coli DH11S carrying pYH31[LB400-bphFGBC] plus pEP51[P6-bphA1/p-o/bphA2] grown for 50 generations as in lane 1. Sizes are indicated on the left in kilodaltons.

Expression in E. coli and in P. putida KT2440 and purification of P6 ht-ISP_BPH. MacKay et al. (29) observed that P6 biphenyl dioxygenase's ISP_BPH component was not detected in induced E. coli DH5 carrying recombinant P6 bphA1 and bphA2 cloned downstream of the lac or T7 promoter. They suggested that the lack of P6 ISP_BPH component in these cells was due to either inefficient translation of bphA1A2 or rapid degradation of the gene product (29).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Absorption spectra of ht-P6 ISPBPH (40 µM) obtained from recombinant P. putida KT2440 cells carrying pEP31[P6-bphA1/p-o/bphA2] (solid line) and obtained from recombinant E. coli DH11S cells carrying pEP31[P6-bphA1/p-o/bphA2] (dashed line).

Expression in E. coli and purification of ht-_B-356P6 and ht-_LB400P6. SDS-polyacrylamide gel electrophoresis (PAGE) of purified preparations of ht-_LB-400P6 and ht-_B-356P6 showed two single peptide bands of M_r corresponding to that of ht-_B-356 or ht-_LB400 with _P6 (Fig. 4, lanes 7 and 8; compared to lane 1 to 3). Both enzymes showed spectral features typical of Rieske-type proteins. When biphenyl was the substrate, ht-_LB-400P6 was very poorly active (only traces of the dihydrodiol metabolites were produced from biphenyl), but as shown below, the enzyme was able to transform various chlorobiphenyls efficiently. In this case, however, two of the four purified preparations that we obtained were very poorly active toward all congeners tested, suggesting that the enzyme becomes easily inactivated during purification. On the other hand, ht-_B-356P6 was stable and catalyzed the hydroxylation of biphenyl at a rate similar to that of ht-_P6P6.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE of purified active preparations of ISPBPH chimeras. Lane 1, P6 ht-ISPBPH, 50 pmol; lane 2, LB400 ISPBPH, 80 pmol; lane 3, B-356 ISPBPH, 50 pmol; lane 4, Mr marker; lane 5, ht-P6LB400, 70 pmol; lane 6, ht-P6B-356, 170 pmol; lane 7, ht-LB400P6, 60 pmol; lane 8, ht-B-356P6, 50 pmol. P6 ht-ISPBPH, ht-P6LB400, and ht-P6B-356 were produced from P. putida KT2440; the other chimeras were produced from E. coli M15. Sizes are indicated on the left in kilodaltons.

Expression in E. coli and in P. putida and purification of ht-_P6LB400 and ht-_P6B-356. Purified preparations of ht-_P6B-356 and ht-_P6LB400 chimeras containing equivalent amounts of each subunit were obtained from E. coli carrying pQE31[P6-bphA1/B-356-bphE] and pQE31[P6-bphA1/LB400-bphE], respectively. However, as for ht-_P6P6 (see above), the purified enzymes were not active and the spectral features of the purified enzyme preparations were not typical of Rieske-type proteins (not shown).

Activities of purified ht-P6 ISP_BPH and chimeras derived from it toward selected chlorobiphenyls. In a previous investigation (17), purified ht-LB400 BPH dox metabolized 2,2'-dichlorobiphenyl efficiently but 3,3'-dichlorobiphenyl poorly, whereas ht-B-356 BPH dox metabolized 3,3'-dichlorobiphenyl but not 2,2'-dichlorobiphenyl efficiently. Furthermore, unlike ht-LB400 BPH dox, ht-B-356 BPH dox was unable to catalyze the meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl. In another investigation (29), resting cell suspensions of recombinant P. putida KT2442 carrying P6 bphA1A2A3A4 did not metabolize 2,2'-dichlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl.

	DISCUSSION

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In a previous work (18), we had reported that purified monomeric B-356 ht-ISP_BPH  subunit produced alone in recombinant E. coli cells was able to assemble an intact [2Fe-2S] Rieske cluster, showing that the  subunit is not involved in folding the Rieske center. However, the purified B-356 ht-ISP_BPH  subunit could not associate in vitro with the purified  subunit to generate an active ₃₃ protein. Conversely, the  subunit combined in cell extract with purified exogenous  subunit to generate an active complex, suggesting the presence in E. coli of a cell constituent that interacts with the  subunit to maintain its correct folding in the absence of the  subunit (18). In the present work, we show that rhodococcal ISP_BPH subunits produced in E. coli cells are assembled into the correct ₃₃ configuration (this was also true for the _P6LB400 and _P6B-356 chimeras). However, the [2Fe-2S] Rieske cluster of the _P6 subunit is incorrectly assembled in E. coli but correctly assembled in Pseudomonas. Altogether these observations show that the subunit association is independent of the Rieske center assembly and suggest that the maturation of the ISP_BPH [2Fe-2S] cluster, like subunit association, may require the involvement of cell constituents such as chaperones.

ht-P6 ISP_BPH is produced active in Pseudomonas cells, showing the importance of choosing the proper organism to express heterologous proteins. Since aryl hydroxylating dioxygenases are found mostly in gram-negative aerobes such as Pseudomonas, Alcaligenes, and Comamonas and in the gram-positive rhodococci, pEP31 and pEP51 constructed during this work provide potentially useful tools to further investigate the biochemical features of these enzymes.

In a recent investigation with B-356 and LB400 ISP_BPH, the catalytic features of _B-356LB400 toward chlorobiphenyls were found to be very similar to those of LB400 BPH dox. On the other hand, with one exception (16), all recent reports (7, 31, 32, 36) indicated that the substrate specificity of the aryl hydroxylating dioxygenase is determined by the terminal oxygenase's  subunit; the  subunit was not found to contribute to this function. The fact that these investigations were done with whole-cell suspensions of recombinant E. coli clones carrying the four genes required for dioxygenase activity on plasmids preclude direct comparison with data obtained with in vitro-reconstituted purified enzyme. Nevertheless, it was imperative to verify if the behavior of the chimera obtained by exchanging the  and  subunits of LB400 and B-356 ISP_BPH was an exception.

Unlike the data obtained with _B-356LB400 (17), none of the four hybrids described in this work showed features identical to those that characterize the parent which provided the  subunit. Interestingly, the catalytic activity toward chlorobiphenyls of _LB400P6 was similar to that of LB400 BPH dox. Based on this result, it would be tempting to conclude that the substrate specificity pattern is determined by the  subunit alone and that _B-356LB400 is an exception. However, it is noteworthy that _LB400P6 was practically inactive on biphenyl. Furthermore, the catalytic features of the three other chimeras studied in this investigation differed significantly from those of the parent which provided the  subunit. Thus, although 2,2'-dichlorobiphenyl is a very poor substrate for B-356 ISP_BPH, _B-356P6 oxygenate this congener faster than 3,3'-dichlorobiphenyl. Furthermore, unlike both parents, _P6LB400 was unable to oxygenate 2,2'-dichlorobiphenyl but showed a slight activity toward 2,2',5,5'-tetrachlorobiphenyl, which was not attacked by P6 ISP_BPH. Together these data support the previous conclusion that each new chimera obtained by exchanging the  or  subunit of parent dioxygenases acquires its own new catalytic features that are not determined exclusively by one or the other subunit (16, 17).

Two structural features are essential to obtain an active enzyme. First, the Rieske cluster must be correctly assembled. Second, we have previously reported that ISP_BPH must associate into ₃₃ heterodimer to be active (18). Based on the present data, it is likely that the three-dimensional structure of the enzyme's catalytic region, as determined by the association between the  and  subunits, will determine the range of substrates that the enzyme can oxygenate. Selected amino acid residues of the  or  subunits are likely, because of their position or charge, to interfere with the enzyme-substrate interaction. However, depending on the final three-dimensional structure of the catalytic region, the amino acid residues that affect the substrate selectivity in one type of - arrangement will not inevitably affect the substrate selectivity in other types of - arrangements. This conceptual model explains why the amino acid residues of the oxygenase  subunit that were found to affect the substrate specificity of strain LB400 and KF707 dioxygenases (22, 30) had no effect on the enzyme reactivity pattern of _B-356B-356 or in _B-356LB400 (17). However, structure analysis of the biphenyl dioxygenase oxygenase component will be required to assess this hypothesis. Recently, Kauppi et al. (21) reported the three-dimensional structure of the terminal oxygenase component of the homologous naphthalene dioxygenase. Structure analysis shows a major involvement of the  subunit in enzyme catalytic activity. However, so far, structure analysis has not helped to identify the role of the  subunit in enzyme catalytic activity or specificity.

Nevertheless, if structural features of the  subunit influence the enzyme's specificity, this fact will be of consequence for enzyme-engineering programs designed to create new enhanced enzymes for the degradation of more persistent chlorobiphenyls. Crameri et al. (9) have recently shown that the use of homologous genes to provide functional diversity accelerates the in vitro-directed evolution process based on DNA shuffling. Kumamaru et al. (23) have shuffled strain LB-400 bphA with P. pseudoalcaligenes KF707 bphA1 and successfully obtained mutants expressing phenotypes of both parents. However, our data suggest that the development by molecular evolution of mutants able to catalyze the oxygenation of congeners that both parents are unable to oxygenate may also have to take into consideration other portions of the ISP_BPH molecule, including perhaps the  subunit.