INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

A fraction of the 209 polychlorinated-biphenyl (PCB) congeners can be transformed into chlorobenzoates by the bacterial biphenyl oxidative catabolic pathway. The pathway involves four enzymatic steps (9, 28). The biphenyl dioxygenase (BPH dox) catalyzes the first reaction of this pathway. Aromatic ring dioxygenases catalyze dihydroxylation reactions on two adjacent carbons of the aromatic ring (23). Some of these enzymes can oxygenate a broad range of substrate analogs. For example, naphthalene dioxygenase can catalyze the hydroxylation of several polycyclic aromatic hydrocarbons (18) and BPH dox can oxygenate various PCB analogs (4, 7, 16). Details about the structural features which are responsible for the enzyme's substrate recognition, binding, and orientation in the direction of the active site will help us to design new enzymes able to use a broader range of substrates than that currently used.

BPH dox has been studied from Comamonas testosteroni B-356 (13, 14) and from Pseudomonas sp. strain LB400 (11, 12). It comprises three components (11, 13, 14). These are the terminal oxygenase, an iron-sulfur protein (ISP_BPH) made up of an  subunit (M_r = 51,000) and a  subunit (M_r = 22,000), a ferredoxin (FER_BPH; M_r = 12,000), and a ferredoxin reductase (RED_BPH; M_r = 43,000). The genes that code for these components in both strain B-356 and strain LB400 are bphA (ISP_BPH  subunit), bphE (ISP_BPH  subunit), bphF (FER_BPH), and bphG (RED_BPH) (6, 31). BPH dox hydroxylates adjacent ortho-meta carbons of one of the biphenyl rings to generate 2,3-dihydro-2,3-dihydroxybiphenyl. FER_BPH and RED_BPH are involved in electron transfer from NADH to ISP_BPH (14). One common feature characterizing the terminal oxygenase components of all aromatic ring-hydroxylating dioxygenases is the presence of a [2Fe-2S] Rieske center located on the  subunit (13, 25, 31), which is believed to be involved in electron transfer from the ferredoxin component to a mononuclear Fe²⁺, which activates molecular oxygen for insertion into the substrate (3, 23). Evidence suggests that the mononuclear Fe²⁺ is coordinated from a domain of the ISP_BPH  subunit (15). Therefore, the  subunit appears to serve a major catalytic function. However, the function of the  subunit in enzyme activity has yet to be elucidated.

Purified active ISP_BPH preparations were obtained from Pseudomonas sp. strain LB400 (11) and from C. testosteroni B-356 (14), whereas B-356 RED_BPH and FER_BPH were purified from Escherichia coli recombinant clones as active His-tagged (H-t) proteins (14). Active preparations of H-t B-356 ISP_BPH were also obtained (13).

Pseudomonas sp. strain LB400 is one of the best-performing PCB-degrading gram-negative bacteria. An important feature that distinguishes strain LB400 from other PCB degraders is its capacity to catalyze the oxygenation of adjacent meta-para carbons of congeners such as 2,2',5,5'-tetrachlorobiphenyl, in which there are no free adjacent ortho-meta carbons (4). Furthermore, strain LB400 metabolizes the double-ortho-substituted congener 2,2'-dichlorobiphenyl very efficiently whereas the double-meta- or -para-substituted congeners such as 3,3'- and 4,4'-dichlorobiphenyl are degraded poorly (4, 7, 10). In spite of the fact that the deduced amino acid sequences of the LB400 and Pseudomonas pseudoalcaligenes KF707 bphA, bphE, bphF, and bphG gene products show 95.5, 99.5, 100, and 100% identity, respectively, KF707 BPH dox is unable to attack 2,2',5,5'-tetrachlorobiphenyl (7, 10, 17). In addition, it poorly metabolizes the double-ortho-substituted congener and, unlike strain LB400, it degrades the double-para-substituted 4,4'-dichlorobiphenyl more efficiently. Results of recent investigations suggest that a relatively small number of amino acid residues of the carboxy-terminal portion of the ISP_BPH  subunit control the substrate selectivity patterns of both strains and their ability to catalyze meta-para dioxygenation (7, 17, 24). However, because the ISP_BPH  subunits of the two strains used in this study differed by a single amino acid residue, it was impossible to determine any involvement of the  subunit in substrate selectivity.

Strain B-356 BPH dox has unique structural features that distinguish it from strain LB400 BPH dox (the amino acid sequences of the LB400 and B-356 ISP_BPH  and  subunits show 76 and 70% identity, respectively) (31). Furthermore, unlike strain LB400, strain B-356 metabolized the double-meta-substituted congener 3,3'-dichlorobiphenyl more efficiently than 2,2'- and 4,4'-dichlorobiphenyl. When tested as a resting-cell preparation in a 1-ml volume, 2.4 mg of the 3.3 mg of 3,3'-dichlorobiphenyl was degraded within 12 h, compared to 0.6 and 0.17 mg of 4,4'- and 2,2'-dichlorobiphenyl, respectively (2). In addition, B-356 was unable to degrade 2,2',5,5'-tetrachlorobiphenyl (2).

Because the recombinant H-t components of B-356 BPH dox have retained all major biochemical features of the parental proteins, affinity chromatography of tagged protein was proposed as a useful tool for examining features of various purified aryl dioxygenase components (13). It especially offers the possibility of comparing the characteristics of purified reconstituted chimeric ISP_BPHs, comprised of  and  subunits derived from distinct parent enzymes. In this work we examine the reactivity pattern of BPH dox toward various PCBs and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISP_BPH  or  subunit of strain LB400 with the corresponding subunit of strain B-356. Results show that the structure of the  subunit influences both the capacity of the enzyme to catalyze the meta-para oxygenation of the substrate and its substrate reactivity pattern toward PCBs.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

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 SG13009 cured of pREP4 (obtained during this work), C. testosteroni B-356 (1), and Pseudomonas sp. strain LB400 (4) (also referred as Burkholderia sp. strain LB400 or Pseudomonas cepacia LB400 [17]). The media used were Luria-Bertani broth (26), H-plate medium (26), and MM30 (29). The plasmids used were pQE31 and pQE51 (Qiagen, Inc.) and pYH31 (to be described elsewhere), which is a new P15A-based plasmid obtained by introducing the operator and promoter region of pQE31, the six-His fusion gene, and the multiple cloning site of pQE31 into the unique HindIII site of pREP4. pHY31 is compatible with ColE1-based plasmids.

Protein characterization. Sodium dodecyl sulfate (SDS)-polyacrylamide gels were developed according to the method of Laemmli (20). Proteins were stained with Coomassie brillant blue (26). Protein concentrations were estimated by the method of Lowry et al. (21) with bovine serum albumin as the standard. The concentrations of H-t ISP_BPH and FER_BPH preparations were also determined spectrophotometrically. The published ₄₅₀ of 10,100 M¹ cm¹ (11) was used to determine the concentration of H-t LB400 ISP_BPH, and the published ₄₅₅ of 8,300 M¹ cm¹ (13) was used to determine the concentrations of H-t B-356 ISP_BPH preparations. The concentrations of both LB400 and B-356 H-t FER_BPH preparations were determined with the ₄₆₀ of 7,455 M¹ cm¹ established for the Rieske center of B-356 FER_BPH (14). The concentrations of _LB400B-356 and _B-356LB400 chimeras were determined with an ₄₅₅ of 8,300 M¹ cm¹. The M_r of native protein was determine by high-performance liquid chromatography (HPLC) as described previously (13).

Monitoring of the enzymes' activities and identification of metabolites. Enzyme assays for BPH dox reactions were performed as described previously (13). A reaction was initiated by adding 100 nmol of biphenyl or of one of the following chlorobiphenyls: 2,2'-, 3,3'-, or 4,4'-dichlorobiphenyl; 2,5-dichlorobiphenyl; and 2,2',5,5'-tetrachlorobiphenyl (all from ULTRAScientific, Kingstown, R.I.) (added in 2 µl of acetone). The reconstituted LB400 H-t BPH dox comprised H-t LB400 ISP_BPH plus H-t LB400 FER_BPH and H-t B-356 RED_BPH; the reconstituted B-356 BPH dox comprised H-t B-356 ISP_BPH plus H-t B-356 FER_BPH and H-t B-356 RED_BPH. Catalytic oxygenation was evaluated by monitoring substrate depletion by HPLC analysis as described previously (13), except that the UV detector of the Hewlett-Packard series 1050 HPLC was set at 203 nm. The K_m and maximal rate of metabolism (V_max) for biphenyl was obtained as described previously (13). The catalytic oxygenation of PCBs was evaluated by monitoring substrate depletion 5 min after initiation of the reaction when 100 nmol of substrate was added to initiate the reaction. The metabolites were detected with a Perkin-Elmer LC95 UV- and visible-light detector set at the maximal wavelength established by Haddock et al. (12). Purification of metabolites was as previously described (13, 30). The 2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase assay was performed as previously described (30). The 3,4-dihydroxybiphenyl used as the standard was from ULTRAScientific. Metabolites were identified by gas chromatographic-mass spectrometric (GC-MS) analysis of their trimethylsilyl (TMS) or butylboronate derivatives, using protocols described previously (14, 27). All values reported in the present study are averages of the results of triplicate experiments for at least two distinct enzyme preparations.

Site-directed mutagenesis at position Thr-375 of the strain B-356 ISP_BPH  subunit. Site-directed mutagenesis was carried out with Pharmacia Biotech's unique site elimination mutagenesis kit, according to the protocol described by Wang and Sul (32). The original sequence of the strain B-356 ISP_BPH  subunit, 5'-GC TTG CAG AAG ATC CGC ACC TTT AAC GCC GGC GGC-3', was modified to 5'-GC TTG CAG AAG ATC CGC AAC TTT AAC GCC GGC GGC-3' by replacing Thr-375 with Asn-375 (boldface). Successful mutations were identified by DNA sequencing. Mutant genes were cloned in pQE31. The mutant enzyme was expressed in E. coli M15 and purified as fused H-t protein according to protocols described previously (13).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Characterization of H-t LB400 ISP_BPH and LB400-B-356 ISP_BPH chimeras. Purified H-t LB400 ISP_BPH was obtained as described in Materials and Methods. SDS-polyacrylamide gel electrophoresis (PAGE) of the purified preparation showed two single peptide bands with M_rs corresponding to those of the H-t  and  subunits of the LB400 ISP_BPH component (Fig. 1A). SDS-PAGE of H-t LB400 FER_BPH preparations showed a single band of the expected M_r (Fig. 1A).

View larger version (53K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of purified preparations of BPH dox components. (A) Lane 1, preparation of H-t LB400 ISPBPH (3 µg); lane 2, Mr markers; lane 3, preparation of H-t LB400 FERBPH (5 µg); (B) lane 1, Mr markers; lane 2, preparation of the H-t B-356LB400 ISPBPH hybrid (2.5 µg); (C) lane 1, Mr markers; lane 2, preparation of the H-t LB400B-356 ISPBPH hybrid (3 µg). Molecular weights (in thousands) are noted at the left sides of the gels.

Reactivities of reconstituted purified H-t LB400 dox and H-t B-356 BPH dox and of H-t LB400-B-356 BPH dox chimeras toward selected PCBs. In a previous study, when biphenyl was used as the substrate, the TMS-derived diol products of the B-356 BPH dox reaction were resolved into two distinct peaks by GC-MS analysis. These two peaks were postulated to be 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl, respectively (14). However, it was later found that when n-butylboronate was used for chemical derivatization of biphenyl metabolites, a single GC-MS peak was obtained. Moreover, the biphenyl-derived dihydrodiol product of the B-356 BPH dox reaction eluted as a single HPLC peak (30). The TMS-derived dihydrodiol obtained from this HPLC peak was resolved as two GC-MS peaks (results not shown). However, when the HPLC-purified 2,3-dihydro-2,3-dihydroxybiphenyl was used as the substrate for the 2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase reaction, both of these TMS-derived dihydrodiol peaks disappeared from the reaction medium and 2,3-dihydroxybiphenyl was the unique metabolite generated in this reaction (results not shown). 3,4-Dihydroxybiphenyl was not produced. Therefore, the resolution of the TMS-derived dihydrodiol into two GC-MS peaks is an artifact. Although at this time we cannot provide an explanation for this artifact, the data presented above clearly show that our former assumption that B-356 BPH dox can catalyze a meta-para hydroxylation of biphenyl was erroneous.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   GC-MS spectra of the butylboronate-derived metabolites obtained from 2,5-dichlorobiphenyl when the substrate was oxygenated with B-356 H-t BPH dox (A), LB400 H-t BPH dox (B), or H-t B-356LB400 BPH dox (C). n-Bu, n-butylboronate.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   GC-MS spectra of the dihydrodiol metabolite produced from 2,2',5,5'-tetrachlorobiphenyl when H-t B-356LB400 ISPBPH was used to reconstitute BPH dox. n-Bu, n-butylboronate.

Site-directed mutagenesis at position Thr-375 of the strain B-356 ISP_BPH  subunit. Mondello et al. (24) have categorized 15 strains into two groups based on their ability to degrade PCB congeners. One group (LB400 type) showed a broad range of congener specificity, including the capacity to degrade 2,2',5,5'-tetrachlorobiphenyl, whereas the second group (KF707 type) showed a much narrower range of PCB congener specificity. Sequence comparison between the ISP_BPH  subunits of these two groups identified four regions of the protein C-terminal portion in which specific sequences were consistently associated with either broad or narrow substrate specificity (24). Strain B-356 fits in with the KF707-type strains. It shows a narrow congener substrate specificity. It poorly degrades 2,2'-dichlorobiphenyl, and it is unable to catalyze the meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl. Furthermore, the amino acid residues of all four regions identified by Mondello et al. (24) as being involved in determining substrate specificity are identical to the residues found in the KF707-type strains (Fig. 4). Moreover, among the residues that are not conserved in all the sequences shown in Fig. 4, 42 residues found in the B-356 ISP_BPH  subunit are also found in the KF707-type ISP_BPH, compared with only 4 residues found in the LB400 type.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Sequence comparison between the C-terminal portion of the B-356 ISPBPH  subunit with those of KF707- and LB400-type ISPBPHs. Regions I, II, III, and IV are those designated by Mondello et al. (24). Residues above the LB400 sequence are, according to the work of Mondello et al. (24), residues found at these positions in other LB400-type ISPBPHs; residues below the KF707 sequence are those found at these positions in other KF707-type ISPBPHs. Dashes represent residues that are conserved in all three sequences. Residues of B-356 ISPBPH in boldface type are found in KF707-type ISPBPHs; those in lightface type are found in LB400-type ISPBPHs. Characters in italics are either found in both types or differ from both types.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study we have engineered novel ISP_BPH hybrids by replacing the B-356 ISP_BPH  or  subunit with the corresponding polypeptide recruited from strain LB400 BPH dox. The ISP_BPH chimeras representing the two associations (the _LB400B-356 and _B-356LB400 chimeras) were functional.

When the catalytic activities towards various PCBs of the novel chimeric enzymes were compared to those of the parent enzymes, results showed that the structure of the ISP_BPH  subunit influences the reactivity pattern of the enzymes as well as their capacity to catalyze the oxygenation of the meta-para carbons. At first glance, these results appear to contradict those obtained by Mondello et al. (24) and Kimura et al. (17), who found that only a small number of amino acid residues located in the carboxy-terminal halves of the KF707 and LB400 ISP_BPH  subunits control the enzymes' reactivities toward PCB congeners. However, it is noteworthy that these studies were carried out with two very closely related dioxygenases. The sequences of the  subunits for LB400 and KF707 ISP_BPH differ by a single amino acid residue (7). In the present study, by using two more distantly related BPH doxes, it was possible to show an influence of the structure of the  subunit on the enzyme reactivity pattern. Furthermore, data obtained with the LB400-B-356 chimeric enzymes show that the amino acid residues of the LB400 and KF707  subunits, which were found in previous investigations to greatly influence the substrate reactivity patterns of LB400 and KF707 BPH dox, did not appear to influence the reactivity patterns of B-356 BPH dox and of LB400-B-356 BPH dox chimeras.

By changing amino acids 335 to 341 (Thr-Phe-Asn-Asn-Ile-Arg-Ile [region III]) of the Pseudomonas sp. strain LB400 ISP_BPH  subunit to the sequence Ala-Ile-Asn-Thr-Ile-Arg-Thr as in the P. pseudoalcaligenes KF707 ISP_BPH  subunit, Erickson and Mondello (7) obtained mutants which were able to efficiently metabolize the otherwise poorly degraded substrate 4,4'-dichlorobiphenyl, thus broadening the enzyme's substrate reactivity.

As noticed above, the region III amino acid sequence of the strain B-356 ISP_BPH  subunit differs from that of strain KF707 by a single amino acid residue (Fig. 4). Yet, unlike the enzyme of strain KF707, both strain B-356 BPH dox and the chimeric enzyme _B-356LB400 ISP_BPH poorly metabolize 4,4'-dichlorobiphenyl.

Similarly, changing simultaneously Asn-377 and Phe-336 of the LB400 ISP_BPH  subunit to Thr-377 and Ile-336 (as in the KF707  subunit) drastically reduced the capacity of the mutant to catalyze the meta-para oxygenation of the molecule (24). Changing Thr-376 of the KF707 ISP_BPH  subunit to Asn-376, as in LB400, resulted in an expansion of the range of biodegradable congeners, including those requiring a meta-para attack. However, although the corresponding position is occupied by a Thr in the strain B-356  subunit, _B-356LB400 ISP_BPH, unlike KF707 ISP_BPH, can transform 2,2',5,5'-tetrachlorobiphenyl into the 3,4-dihydro-3,4-dihydroxychlorobiphenyl derivative. Furthermore, changing Thr-375 of the B-356 ISP_BPH  subunit to Asn-375, as in LB400, did not confer to the mutant the capacity to oxygenate 2,5-dichlorobiphenyl onto meta-para carbons.

Finally, the fact that the substrate selectivity pattern of the _LB400B-356 chimera differs from that of LB400 and of B-356 BPH dox is further evidence that the structures of both subunits influence the substrate selectivity of the enzyme.

Our data are insufficient to precisely determine the function of the  subunit in BPH dox activity. However, for the first time, we provide clear evidence with purified enzyme preparations that both the  and  subunits of the aryl-hydroxylating dioxygenases influence the enzyme-substrate interaction. The amino acid residues of the  subunit that affect enzyme reactivity in one type of - arrangements, that of the _LB400LB400 arrangement, have no effect on other types of - arrangements, such as that of the _B-356LB400 chimera. To explain these results, it is likely that a catalytic poach is created by the association between the  and  subunits. Structural features of both subunits would then influence the dimension and shape of the catalytic poach to determine which PCBs can be oxygenated as well as the orientations of the adjacent reactive carbons toward the active site. This observation is important in terms of engineering enzymes to increase the range of the catalytic activities toward PCBs. Current investigation in our laboratory aims at identifying the residues of both the large and small ISP_BPH subunits which are involved subunit association as well as substrate binding and orientation in the direction of the enzyme's active site.