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J. Biol. Chem., Vol. 279, Issue 46, 47480-47488, November 12, 2004

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Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III^*

Diane Barriault and Michel Sylvestre

From the Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, 245 Boulevard Hymus, Pointe-Claire, Québec H9R 1G6, Canada

Received for publication, June 17, 2004 , and in revised form, August 19, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) 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 subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr³³⁵-Phe³³⁶-Ile³³⁸-Ile³⁴¹) 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 Phe³³⁶ with Met or Ile with a concomitant change of Thr³³⁵ 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 Thr³³⁵-Phe³³⁶ with Ala³³⁵-Leu³³⁶ did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala³³⁵-Met³³⁶ 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 Ala³³⁵-Met³³⁶ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Biphenyl dioxygenase (BPDO)¹ catalyzes the first reaction of the biphenyl catabolic pathway. BPDO comprises three components (1, 2): The iron-sulfur oxygenase (ISP_BPH) made up of an subunit (M_r = 51,000) and a subunit (M_r = 22,000), the ferredoxin (FER_BPH, M_r = 12,000), and the ferredoxin reductase (RED_BPH, M_r = 43,000). The encoding genes for Burkholderia xenovorans LB400 (3), which is also called Burkholderia (Pseudomonas) sp. LB400 (4, 5), are bphA (ISP_BPH subunit), bphE (ISP_BPH subunit), bphF (FER_BPH), and bphG (RED_BPH). BPDO can oxygenate several polychlorinated biphenyl (PCB) congeners. The application of BPDO for efficient PCB degrading processes will require an expansion of the range of PCB substrates it can oxygenate. The catalytic oxygenation of biphenyl occurs normally on carbons 2 and 3 to generate the cis-2,3-dihydro-2,3-dihydroxybiphenyl, which is dehydrogenated by the cis-2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase encoded by bphB in strain LB400. The resulting catechol 2,3-dihydroxybiphenyl is then cleaved by the 2,3-dihydroxybiphenyl 1,2-dioxygenase encoded by bphC in strain LB400. 2-Hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid (HOPDA) is then hydrolyzed by the HOPDA hydrolase (BphD) to yield benzoate and 2-hydroxypentanoic acid (Fig. 1).

View larger version (12K): [in this window] [in a new window]   FIG. 1. PCB degradation pathway of B. xenovorans LB400 showing the genes encoding the enzymes involved in each catalytic step.

In addition to their influence on the range of chlorobiphenyls used as substrate by the enzyme, amino acids of region III also appear to be implicated in regiospecificity toward 2,2'-CB. Thus Suenaga et al. (12) found that replacing Ile³³⁶ of KF707 BphA with Phe as in LB400 BphA changed the regiospecificity toward 2,2'-dichlorobiphenyl (2,2'-CB) to favor production of 2,3-dihydroxy-2'-chlorobiphenyl.

The ortho-substituted PCB congeners such as 2,2'-, 2,6-, and 2,6,2',6'-CB are among the most resistant to microbial attack. LB400 BPDO is among the few BPDO of natural occurrence that can oxygenate 2,2'-CB efficiently (9). In previous work we found that evolved BPDOs selected for their ability to oxygenate 2,2'-CB at a higher rate than the parental LB400 BPDO were able to catalyze a broader range of PCB congeners than LB400 BPDO (6). Although the amino acid residues of region III BphA influence greatly the range of PCB congeners oxygenated by the enzyme, based on the alignment of sequences of BphAs that are found in data bases, the diversity of sequence patterns of this stretch of amino acids is rather limited (6). In this work, we have used an approach that combines a PCR-based method of random mutagenesis at targeted multi-sites plus DNA shuffling to create a library of BPDO variants that were randomly mutated simultaneously on four residues Thr³³⁵-Phe³³⁶-Asn³³⁸-Ile³⁴¹ of region of LB400 BphA. The purpose of the work was to evaluate the extent of variation that can occur in region III of BphA of LB400 with no loss of activity toward 2,2'-CB and the influence of new sequence patterns on catalytic activity toward this congener and other chlorobiphenyls. One of the most potent variant of the library (variant p4) exhibited a higher turnover rate of oxygenation of 2,2'-CB, but its regiospecificity toward this congener was altered. The rates of metabolism and the metabolite profiles of this variant toward a range of chlorobiphenyl congeners were compared with those obtained when LB400 BPDO catalyzed the reaction. The data suggest that despite the fact that residues 335 and 336 exert a strong influence on the regiospecificity toward 2,2'-CB and on the turnover rate toward several congeners, the pattern of chlorine substitution on the PCB substrate imposes its orientation toward the catalytic active center.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains, Plasmids, and General Protocols—Escherichia coli DH11S (13) was used in this study. Plasmid pQE31[LB-400-bphAE], in which bphA was mutated to introduce an AvrII site at position 1354, was described previously (14); it encodes for a fused His-tagged ISP_BPH (14, 15) carrying the His tag on BphA. Plasmid pYH31[LB400-bphF-GBC] was described previously (16). This plasmid encodes for the ferredoxin (BphF) and the ferredoxin reductase (BphG) components of LB400 BPDO and for 2,3-dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (BphB) and 2,3-dihydroxybiphenyl 2,3-dioxygenase (BphC) from B. xenovorans LB400, which are the second and third enzymes of the biphenyl catabolic pathway. The coupled reaction involving BPDO plus BphB and BphC transforms biphenyl into its yellow HOPDA.

To construct pDB31[LB400-bphFG], bphFG was amplified as a 1555-bp BamHI/KpnI fragment from LB400 DNA using antisense primers 5'-GCGGGATCCTATGAAATTTACCAGAGTTTG-3' and 5'-CTGGTACCGCTTCACCTTTCA-3' and then cloned into the BamHI/KpnI-digested pQE31. DNA general protocols were done according to Sambrook et al. (17). DNA was sequenced by DNA Landmark (St-Jean, Canada).

Preparation of DNA and PCRs to Evolve BPDO and Screening Protocol—Seven degenerated primers, each of which contained degenerated nucleosides at positions to be mutated, were used with appropriate antisense primers to amplify fragments of LB400-bphAE that overlapped over the stretch of DNA that encode for region III (Fig. 2). Primers 1 and 2 were used in conjunction with the reverse primer 3 to amplify the 1065-bp fragments A and B that corresponded to the right end portion of bphA plus bphE. Primer 1 and 2 were degenerated at bp positions 1021, 1022, and 1023 to introduce a mutation to replace Ile³⁴¹ of the protein by any other amino acid. In addition, primer 1 was degenerated at position 1008, and primer 2 was degenerated at bp position 1007. The base pairs at positions 1007 and 1008 belong to the codon encoding amino acid 336 (Phe) of the protein. Fragment A and B extended from the beginning of the DNA stretch that encoded for region III of BphA (sequence Thr³³⁵-Phe³³⁶-Asn³³⁷-Asn³³⁸-Ile³³⁹-Arg³⁴⁰-Ile³⁴¹) to the end of the gene. The five other primers, primers 4, 5, 6, 7, and 8, containing degenerated nucleotides were used in conjunction with primer 9 to amplify a 1023-bp fragment that corresponded to the left end portion of bphA. These five sets of primers amplified DNA fragments C, D, E, F, and G that extended from the beginning of bphA to bp position 1023, which corresponded to the end of region III of BphA (Fig. 2). Therefore fragments C, D, E, F, and G overlapped with fragment A and B inside the DNA stretch that encoded region III. Primer 4 was degenerated at bp positions 1006, 1007, and 1008 to mutate Phe³³⁶ of the protein; primer 5 was degenerated at bp positions 1003, 1004, and 1005 to mutate Thr³³⁵ of the protein; primer 6 was degenerated at bp positions 1012, 1013, and 1014 to mutate Asn³³⁸ of the protein; primer 7 was degenerated at bp positions 1003, 1005, 1006, and 1008 to mutate Thr³³⁵ and Phe³³⁶ together; and primer 8 was degenerated at bp positions 1006, 1008, 1011, and 1014 to mutate Phe³³⁶ and Asn³³⁸ together.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Degenerated primers used to introduce random mutagenesis at multiple sites inside region III. At top, the portion of nucleic acid sequence of LB400 bphAE showing the coding region and the amino acid sequence of region III is shown. The letters on the left designate the amplified fragments; the numbers in squares designate the primers. N represents A, T, G, or C; H represents A, T, or C; D represents A, T, or G; V represents A, C, or G; R represents A or G.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Strategy to evolve BphA. LB400 bphA is drawn at the top showing the position of region III, the Rieske Center, and the mononuclear iron. The degenerated primers (small arrows) described in Fig. 1 were used to amplify families of fragments of bphA that overlapped inside region III. The fragments were digested with DNase I and reassembled by primerless PCR. The PCR product was then amplified with primers 10 and 11 to generate a library of 1238-bp DNA stretches. These were digested with MluI/AvrII, and the resulting library of 864-bp MluI/AvrII fragments was used to replace the corresponding fragment in pQE31[LB400-bphAE].

Whole Cell Assays to Evaluate the PCB Degrading Potency—E. coli DH11S pYH31[LB400-bphFGBC] + pQE31[bphAE] cells expressing variant BphAs were tested for their ability to degrade a synthetic mix of 18 PCBs using a protocol published previously (6). The synthetic mix comprised 1 µM of each of the following congeners: 2,6-CB, 2,4,3'-CB, 2,4,4'-CB, 2,3,4'-CB, 2,6,2',6'-CB, 2,5,2',5'-CB, 2,3,2',3'-CB, 2,4,2',4'-CB, 2,4,3',4'-CB, 3,4,3',4'-CB, 3,4,3',5'-CB, 2,3,4,2',5'-CB, 2,4,5,3',4'-CB, 2,4,5,2',4',5'-CB, 2,3,4,5,2',5'-CB, and 5 µM of 3,3'-CB and 4,4'-CB. The internal standard 2,3,4,5,6,2',3',5',6'-CB, presumed to be not degraded, was present at a concentration of 1 µM. The chlorobiphenyls were obtained from ULTRAScientific (Kingstown, RI). Each experiment included control cultures of E. coli [pYH31bphFGBC] + [pQE31] that were run under conditions identical to the experimental cultures. All of the values reported in the present study are averages from triplicate experiments.

Assays to Identify the Metabolites and Quantify the Catalytic Activity—Metabolites were analyzed from suspensions of IPTG-induced whole cells of E. coli [pDB31bphFG] + [pQE31bphAE]. In this case, log phase cells grown in LB broth were induced for 2 h with 0.5 mM IPTG and then washed and suspended to an optical density at 600 nm of 2.0 in M9 medium (17) containing 0.5 mM IPTG. The cell suspension was distributed by portions of 2 ml in 7-ml glass tubes covered with Teflonlined screw caps. Each tube received 10 µl of a 50 mM acetone solution of the appropriate chlorobiphenyl substrate. They were incubated for 18 h at 37 °C with shaking. Cell suspensions were extracted at neutral pH with ethyl acetate. The metabolites were identified by gas chromatography-mass spectrometry (GC-MS) analyses of their butylboronate or trimethylsilyl derivatives (6, 19). In some cases, the dihydro-dihydroxy metabolites were oxidized by IPTG-induced cell suspensions of E. coli [pQE31 bphB] (19) following a protocol similar to the one described above.

In some experiments, the metabolites were analyzed from catalytic oxygenation using reconstituted BPDO prepared from His-tagged purified enzymes components that were obtained following protocols published previously (2). Catalytic activities were determined from measurement of substrate depletion recorded by GC-MS analysis (15).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Screening for BPDO Variants Able to Oxygenate 2,2'-CB—To randomly mutate targeted residues of region III simultaneously, we used the approach described under "Experimental Procedures" and summarized in Fig. 3. A library of E. coli transformants carrying pQE31[bphAE] plus pYH31[bphFGBC] was generated. Transformants were grown on nitrocellulose membranes and exposed to the vapors of a chlorobiphenyl. The screening assay was based on the production of yellow chloro-HOPDA by these transformants. In a first set of experiments, we found that 20% of the transformants were able to oxygenate the easily oxidizable 4-CB; 50% of them were unable to transform 2,2'-CB into the yellow chloro-HOPDA. Therefore approximately half of the variants that remained active toward 4-CB contained (a) mutation(s) that caused a loss of activity toward 2,2'-CB.

70,000 colonies of the library were exposed to 2,2'-CB vapors. Among the 7,000 yellow-colored colonies, only seven exhibited a clearly more intense color than control colonies expressing parental LB400 BPDO. Based on the assumption that variant exhibiting a higher turnover rate of reaction toward 2,2'-CB would produce larger amount of chloro-HOPDA from 2,2'-CB, we retained these colonies for further examination.

We also noticed that a fraction of the 70,000 transformants examined exhibited a pinkish gray color rather than the expected yellow color when exposed to 2,2'-CB. The latter were presumed to generate a metabolite that BphC was unable to cleave. Therefore, these variants were expected to have changed their regiospecificity toward 2,2'-CB because it has been established that catalytic oxygenation of this congener by LB400 BPDO produces as major metabolite 2,3-dihydroxy-2'-chlorobiphenyl (20, 21) that can be cleaved by BphC. These pinkish gray variants were also characterized.

All of the variants retained were examined for their capacity to degrade a mixture of 18 PCB congeners. Based on previous reports (20, 22–24), with the exception of 2,5,2',5'-CB and 2,3,2',3'-CB, all chlorobiphenyls of the mix are metabolized at a low rate by purified preparations of LB400 BPDO or by E. coli clones expressing this enzyme. For this reason, under our experimental conditions the depletion levels of these congeners by IPTG-induced E. coli cells expressing LB400 BPDO was lower than 20% after 18 h of incubation. These levels of depletion were too low to report statistically significant variations and were therefore not reported precisely (Table I). The mix of 18 congeners did not contain 2,2'-CB because it was not discriminated from 2,6-CB by our GC-ECD chromatography setting, but its degradation was demonstrated in other experiments (see below).

Characterization of the Pinkish Gray-colored Variants—Approximately 50 of the 70,000 clones examined for their ability to convert 2,2'-CB into chloro-HOPDA exhibited a pinkish gray coloration. Seven were sequenced. Variant p1 was a triple mutant in which Thr³³⁵-Phe³³⁶-Ile³⁴¹ was replaced by Ala³³⁵-Leu³³⁶-Val³⁴¹. Variant p8 was a double mutant where Thr³³⁵-Phe³³⁶ was replaced by Ala³³⁵-Ile³³⁶. It is noteworthy that of the seven variants, five (p2, p3, p4, p5, and p7) were double mutants that shared an identical amino acid sequence pattern where Thr³³⁵ was replaced by Ala and Phe³³⁶ was replaced by Met.

Induced E. coli cell suspension expressing p1 BPDO metabolized 2,2'-CB at a rate of 1.5 nmol/h, which was similar to that of cells expressing LB400 BPDO. The rate of depletion of 2,2'-CB by cells expressing p8 BPDO was, however, much lower (0.38 nmol/h). Assays measuring the rate of depletion of 2,2'-CB by His-tagged purified preparations of p4 variant (Ala³³⁵-Met³³⁶) revealed that its specific activity toward 2,2'-CB was in the range of 70–90 nmol 2,2'-CB oxidized per min per mg enzyme compared with values of 35–55 nmol/min/mg enzyme for LB400 BPDO. The specific activity of purified preparations of p4 BPDO was significantly higher than that of LB400 when biphenyl was the substrate. Values of 350–400 nmol biphenyl converted per min per mg p4 BPDO were obtained, compared with 120–150 nmol/min/mg LB400 BPDO. Therefore, the turnover rate of oxidation of biphenyl had increased significantly for this enzyme. It was also the case for several other PCB congeners that were oxygenated more efficiently by E. coli cells expressing p4 BPDO than those expressing LB400 BPDO (Table I). On the other hand, cells expressing p8 BPDO oxygenated 3,3'-CB more efficiently than those expressing LB400 BPDO, but overall, the range of chlorobiphenyls degraded was much smaller than for variant p1 and p4.

Metabolism of 2,2'-CB by BPDO Variants—Haddock et al. (20) reported that two metabolites were generated from oxygenation of 2,2'-CB by LB400 BPDO. The major one, representing about 90% of the product, was identified as 2,3-dihydroxy-2'-chlorobiphenyl. In a recent report (29) we identified the less abundant metabolite as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl.

When the pinkish gray variant p4 was used to catalyze the reaction, the ratio of the metabolites produced from 2,2'-CB differed significantly from the ratio obtained with LB400 BPDO. E. coli cells expressing p4 BPDO produced 30–40% of 2,3-dihydroxy-2'-chlorobiphenyl and 60–70% of cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB (Table II). In addition, there was a small amount (1–5%) of a dihydroxy-dichlorobiphenyl, which was confirmed to be 3,4-dihydroxy-2,2'-dichlorobiphenyl (29).

Metabolism of Other PCB Congeners by Variant p4 BPDO— Table III and Fig. 4 show the metabolite profile obtained from selected chlorobiphenyls when LB400 and p4 BPDOs were used to catalyze the oxygenation. The metabolites were identified by GC-MS analysis in comparison with the available data reported in the literature. The table also shows the relative rate of oxygenation of these congeners by p4 BPDO compared with that of LB400 BPDO.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Scheme comparing the regiospecificity of oxygenation of various chlorobiphenyls by LB400 and p4 BPDOs. The arrows show the vicinal carbons that most frequently interact with the activate dioxygen during catalytic reaction. For clarity, the minor sites of oxygenation are not shown.

Similarly, the mutation that occurred in p4 BPDO did not change the regiospecificity toward 2,4,3'-CB, 2,3,4'-CB, 2,4,4'-CB, and 2,4,2',4'-CB, but the rate of conversion of these congeners was faster when p4 was used to catalyze the reaction (Table III). Only one metabolite was produced from 2, 4,2',4'-CB by both enzymes. Based on previous report (24) it was identified as 2,3-dihydroxy-4,2',4'-trichlorobiphenyl. Two metabolites were produced from 2,4,4'-CB. Its trichlorinated dihydro-dihydroxy derivative could not be identified precisely. However, based on the previous report (24) its dichlorinated derivative was identified as 2,3-dihydroxy-4,4'-dichlorobiphenyl, showing that a portion of the substrate was oxygenated on the ortho-meta carbons of the 2,4-chlorinated ring. Unlike 2,4,4'-CB neither LB400 nor p4 BPDO oxygenated 2,4,3'-CB on carbons 2 and 3 of the 2,4-chlorinated ring. Both enzymes generated two trichlorinated dihydro-dihydroxy metabolites that could not be identified precisely from available data. However, the metabolite profiles were identical whether the oxygenation was catalyzed by LB400 or p4 BPDO. Two trichlorinated dihydro-dihydroxy metabolites were also obtained from 2,3,4'-CB. Their precise identity was not determined, but an identical metabolite profile was obtained for both enzymes.

The metabolite profile and rate of metabolism of the orthometa congeners 2,5,2',5'-CB, 2,3,2',3'-CB and 2,3,4,2',5'CB were the same for both enzymes (Table III) as well as their rate of metabolism. Based on previous reports (22, 24), 2,5,2',5'-CB was oxygenated on carbons 3 and 4 to generate cis-3,4-dihydro-3,4-dihydroxy-2,5,2',5'-tetrachlorobiphenyl. The metabolites produced from 2,3,2',3'-CB by LB400 BPDO have been identified recently (29) as being cis-4,5-dihydro-4,5-dihydroxy-2,3,2'3'-tetrachlorobiphenyl and cis-5,6-dihydro-5,6-dihydroxy-2,3,2'3'-tetrachlorobiphenyl produced in a ratio of 90:10, showing that oxygenation on the vicinal meta-para carbons 4 and 5 is largely favored (Fig. 4). Based on GC-MS analysis of their butylboronate derivatives, the same two metabolites were produced in same ratio by p4 BPDO (Table III). Similar to previous observations (24) that 2,3,4,2',5'CB was metabolized very slowly by LB400 BPDO, the rate of metabolism was not significantly higher when p4 was used to catalyze its oxygenation. (Table III). A single metabolite was detected, and it corresponded to a pentachlorinated dihydro-dihydroxybiphenyl. Based on the fact that 2,5,2',5'-CB and 2,3,2',3'-CB (20, 29) are oxygenated principally on meta-para carbons, the pentachlorinated metabolite is likely to be cis-3,4-dihydro-3,4-dihydroxy-2,3,4,2',5'-pentachlorobiphenyl, but this needs to be confirmed.

The co-planar congener 3,4,3',5'-CB and the mono-ortho coplanar 2,4,3',4'-CB are not oxygenated by purified preparations of LB400 BPDO or E. coli cells expressing the enzyme. A single metabolite identified as a trichlorinated dihydroxybiphenyl was detected when IPTG-induced E. coli cells expressing p4 BPDO were used to catalyze the oxygenation of 3,4,3',5'-CB. It is, however, noteworthy that this metabolite was only detected when the substrate was added at a concentration lower than 10 µM in the culture medium. Bedard et al. (26) have previously reported the fact that the more recalcitrant PCB congeners are degraded more efficiently when present at very low concentration. There is no clear explanation for this observation; however, substrate inhibition of BPDO reaction caused by uncoupling of NADH oxidation to substrate oxidation has been demonstrated for several chlorobiphenyls (27). Nevertheless, clearly, variant p4 has acquired the capacity to oxygenate this co-planar congener. Although the exact position of attack could not be determined, it is noteworthy that the oxygenation does not occur on the two chlorine-free ortho-meta carbons 5 and 6. Otherwise, the metabolite would have been tetrachlorinated.

IPTG-induced E. coli cells expressing p4 BPDOs produced a single metabolite from 2,4,3',4'-CB in a small amount (representing a conversion yield of 1–5% of the substrate). The mass spectral features of its butylboronate derivative were those of a tetrachlorinated dihydro-dihydroxybiphenyl (Table III). However, we were unable to determine whether the oxygenation occurred on the 2,4- or 3,4-chlorinated ring.

Similar to previous congeners, no metabolites were detected when purified LB400 BPDO or E. coli clones expressing this enzyme were used to catalyze the oxygenation of 2,6-CB, confirming data previously reported (6, 23). Variant p4 produced three metabolites from 2,6-CB (Table III). These include the same two dichlorinated dihydro-dihydroxy metabolites that were generated by variant II-9 described previously (6) plus one monochlorinated dihydroxybiphenyl. Although the precise identity of the two dichlorinated dihydro-dihydroxy metabolites could not be determined, the fact that the dihydroxymonochlorobiphenyl metabolite represented 25% of 2,6-CB metabolites showed that variant p4 BPDO oxygenated a non-negligible portion of 2,6-CB on vicinal ortho-meta carbons of the chlorinated ring. This was unexpected based on the observation that p4 did not prefer an ortho-meta oxygenation on the chlorinated side of the 2,2-CB ring. The data suggest that structural factors that influence the regiospecificity toward 2,2'-CB are not the same as those that determine the regiospecificity toward the homologous congener 2,6-CB.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this work we have used the in vitro molecular evolution approach to examine the contribution to regiospecificity and substrate turnover of amino acid residues of BPDO subunit region III. Screening was set up to select for variants exhibiting increased turnover rate or modified regiospecificity toward 2,2'-CB. When compared with LB400 BPDO, several of the selected variants had increased their rates of metabolism of several PCB congeners. In vitro molecular evolution is a very powerful approach to identify structural features associated to selected phenotypes. Many variants selected on the basis of the intensity of the yellow coloration produced from 2,2'-CB were similar or identical to those obtained previously (9) by exchanging residues of LB400 BphA by those of KF707 BphA1. The data suggest that the number of amino acid combinations of region III leading to the increased rate of oxygenation of 2,2'-CB accompanied by no change of regiospecificity are rather limited. The diversity of sequence patterns of region III of variants exhibiting a change in regiospecificity toward 2,2'-CB was also low, but two of the three sequence patterns analyzed conferred an increase of the turnover rate of oxidation toward a range of chlorobiphenyls.

Engineering BPDOs to catalyze the oxygenation of PCBs is a formidable task. The enzyme has to learn to catalyze the oxygenation of an array of structurally distinct compounds, which is determined by the position of their chlorine substitutes. Thus some congeners can take a co-planar configuration; others, the ones that are doubly ortho-substituted on the same ring, can never be co-planar. Regiospecificity toward each chlorobiphenyl congener is directed by its chlorine substitution pattern, through interaction between the chlorine atoms and specific amino acid residues of the protein. The identity of these amino acid residues and their interactions with various chlorobiphenyl congeners is still unknown. However, several amino acids of the C-terminal portion of BphA have already been identified as being critical for enzyme turnover rate and regiospecificity toward selected chlorobiphenyl congeners. Among them, the amino acids of region III of BphA and especially Thr³³⁵-Phe³³⁶-Ile³⁴¹ of LB400 BPDO have been found to influence the substrate turnover rate toward several congeners and the regiospecificity toward 2,2'-CB (6, 9). Changing these critical amino acids alters the orientation that 2,2'-CB occupies inside the catalytic pocket, and in addition it can most likely change the conformation of the biphenyl ring.

Supportive evidence provided by this work and a previous study (12) shows that residue 335 and especially residue 336 are directly implicated in enzyme turnover rates and regiospecificity. Replacing Phe³³⁶ by Met or Ile as in p4 (Ala³³⁵-Met³³⁶) or p8 (Ala³³⁵-Ile³³⁶) variants resulted in a change in regiospecificity toward 2,2'-CB. Suenaga et al. (12) showed that a variant of KF707 BPDO in which Ile³³⁵ (corresponding to Phe³³⁶ of LB400 BPDO) was replaced by Phe changed its regiospecificity toward 2,2'-CB. Furthermore, most of the yellow variants of our collection that catalyzed a predominantly 2,3 oxygenation had retained Phe³³⁶, except y10 (Ala³³⁵-Leu³³⁶). Thus the residue occupying position 336 of LB400 BPDO influences directly the regiospecificity toward 2,2'-CB, and Phe seems to play a significant role in this matter. Based on crystallographic analysis of naphthalene dioxygenase, Suenaga et al. (12) have developed a three-dimensional model of KF707 BphA1. Their model suggested that Ile³³⁵ of KF707 BPDO, which corresponds to Phe³³⁶ of LB400 BPDO, is very close to the active iron center. This residue as well as its immediate neighbors is thus likely to play an essential role in enzyme activity. The mechanism by which these residues interact with the substrate to influence access and orientation of the substrate toward the catalytic site is still unknown. However, data show that the amino acids that are found at these positions in wild-type enzymes are not those that provide the optimal structural features to metabolize an extended range of PCB congeners efficiently; Ala³³⁵-Met³³⁶ variants were in this respect the most active.

There were major differences between variants p4 (Ala³³⁵-Met³³⁶) and p8 (Ala³³⁵-Ile³³⁶) with respect to their turnover rates of oxidation toward chlorobiphenyls. Variant p8 metabolized the para-substituted congeners poorly. The PCB degrading potency of variants exhibiting the Ala³³⁵-Ile³³⁶-Ile³⁴¹ sequence pattern has never been examined before. Mondello et al. (9) have examined the PCB degrading potency of a variant exhibiting an Ala³³⁵-Ile³³⁶-Thr³⁴¹ sequence pattern. This variant had acquired the capacity to oxygenate 4,4'-CB and other doubly para-substituted congeners. Based on these results, 341 in addition to residues 335 and 336 would be able to modulate the turnover rate of oxygenation of a range of chlorobiphenyls. Furthermore, comparison of the regiospecificity toward 2,2'-CB of variant y10 (Ala³³⁵-Leu³³⁶-Ile³⁴¹) with that of variant p1 (Ala³³⁵-Leu³³⁶-Val³⁴¹) further substantiate that residue 341 influences this enzyme feature.

The pinkish gray variants had changed their regiospecificity toward 2,2'-CB favoring oxygenation of carbons 3 and 4. At this time it is not clear why no 5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was detected among the metabolites produced by 2,2'-dichlorobiphenyl because typically BPDO prefers ortho-meta dioxygenation of the phenyl ring. Although the catalytic activity toward a range of PCB congeners was increased considerably in p4, including doubly ortho-substituted and co-planar chlorobiphenyls, it is quite interesting that the regiospecificity toward congeners other than 2,2'-CB was the same for both p4 and LB400 BPDO. It is especially interesting to point out that despite the fact that the chlorinated side of the biphenyl ring was not the preferred site of attack of 2,2'-CB by p4 BPDO, the single metabolite obtained from 2,4,2',4'-CB was the same as the one obtained when LB400 BPDO catalyzed the reaction, and it was identified as a trichlorinated dihydroxybiphenyl. On the other hand, 2,3,2',3'-CB was oxygenated principally on the meta-para carbons by LB400 BPDO. The mutation that occurred in p4 did not alter the regiospecificity of the enzyme toward this substrate. It is noteworthy that the two symmetrical ortho-meta-substituted congeners 2,5,2',5'-CB and 2,3,2',3'-CB are both oxygenated principally on meta-para carbons by both LB400 and p4 BPDO. Altogether, data suggest that despite the fact that residues 335 and 336 exert a strong influence on the regiospecificity toward 2,2'-CB and on the turnover rate toward several congeners, the pattern of chlorine substitution on the PCB substrate imposes its orientation toward the catalytic active center. We will need to wait for the crystal structure of the enzyme to understand how the residues of region III interact with individual congeners to influence their rate of oxygenation and, for some of them, their site of oxygenation. However, by analogy with cytochrome P450_cam (28), it is likely that hydrophobic interactions or hydrogen bonding between the chlorine substitutes on the biphenyl skeleton and specific amino acid residues of the protein influence the orientation of the biphenyl ring inside the catalytic pocket. The identification of the amino acid residues involved in these interactions are mandatory to design strategies to broaden the range of PCB congeners that the enzyme can oxygenate efficiently.

	FOOTNOTES

 
^* This work was supported by Natural Sciences and Engineering Research Council Grants 224153 and 257566. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 514-630-8829; Fax: 514-630-8850; E-mail: Michel.Sylvestre{at}inrs-iaf.uquebec.ca.

¹ The abbreviations used are: BPDO, biphenyl dioxygenase; PCB, polychlorinated biphenyl; 4-CB, 4-chlorobiphenyl; 2,2'-CB, 2,2'-dichlorobiphenyl; 3,3'-CB, 3,3'-dichlorobiphenyl; 4,4'-CB, 4,4'-dichlorobiphenyl; 2,6-CB, 2,6-dichlorobiphenyl; 2,4,3'-CB, 2,4,3'-trichlorobiphenyl; 2,4,4'-CB, 2,4,4'-trichlorobiphenyl; 2,3,4'-CB, 2,3,4'-trichlorobiphenyl; 2,6,2',6'-CB, 2,6,2',6'-tetrachlorobiphenyl; 2,5,2',5'-CB, 2,5,2',5'-tetrachlorobiphenyl; 2,3,2',3'-CB, 2,3,2'3'-tetrachlorobiphenyl; 2,4,2',4'-CB, 2,4,2',4'-tetrachlorobiphenyl; 2,4,3',4'-CB, 2,4,3',4'-tetrachlorobiphenyl; 3,4,3',4'-CB, 3,4,3,4'-tetrachlorobiphenyl; 3,4,3',5'-CB, 3,4,3'5'-tetrachlorobiphenyl; 2,3,4,2',5'-CB, 2,3,4,2',5'-pentachlorobiphenyl; 2,4,5,3',4'-CB, 2,4,5,3',4'-pentachlorobiphenyl; 2,4,5,2',4',5'-CB, 2,4,5,2',4',5'-hexachlorobiphenyl; 2,3,4,5,2',5'-CB, 2,3,4,5,2',5'-hexachlorobiphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid; IPTG, isopropyl -D-thiogalactopyranoside; GC-MS, gas chromatography-mass spectrometry.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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