German Research Centre for Biotechnology (GBF), Division of Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany1
Author for correspondence: Bernd Hofer. Tel: +49 531 6181467. Fax: +49 531 6181411. e-mail: bho{at}gbf.de
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ABSTRACT |
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Keywords: aerobic PCB catabolism, biphenyl dioxygenase, chlorobiphenyls, heterologous gene expression, substrate-binding site
Abbreviations: ARHDO, aromatic-ring-hydroxylating dioxygenase; BDO, biphenyl dioxygenase; CB, chlorobiphenyl; OE, overlap extension; PCB, polychlorobiphenyl
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INTRODUCTION |
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METHODS |
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Oligonucleotides.
These were purchased from Life Technologies and are listed in Table 1.
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Construction of chimaeric genes.
Hybrid genes were obtained by recombinant DNA techniques which were carried out as described previously (Sambrook et al., 1989 ; Hofer et al., 1994
), unless otherwise indicated. Hybrids bphA1-H01 and -H02 were constructed by replacement in pAIA50 of the bphA1 MluIAgeI or MluIAscI fragment of strain LB400 by the respective fragment of the bphA1 gene from strain P6. bphA1-P6 fragments were synthesized by PCR (Saiki et al., 1988
). Appropriate primer pairs (LP1/LP2 or LP1/LP4, respectively) introduced the required restriction sites at their ends. The resulting plasmids were designated pAIA501 and pAIA502. bphA1-H04 was constructed as bphA1-H02, with the modification that the 3'-terminal PCR primer (LP10) introduced a stop codon (TGA) behind codon 452 of bphA1-P6. This yielded pAIA504. bphA1-H03 was constructed like bphA1-H01, with the modification that the newly inserted fragment was an LB400/P6 hybrid. This was obtained by separate PCR syntheses of the respective P6 and LB400 fragments, their purification by agarose gel electrophoresis and fusion by overlap extension (OE) PCR (Higuchi et al., 1988
). The first PCR product was derived from pAIA502, using primers LP5 and LP6; the second PCR product was derived from pAIA50, using primers LP7 and LP8; both PCR products were joined using primers LP5 and LP8. Cloning of the MluIAgeI fragment of this product yielded pAIA503. The construction of bphA1-H09 involved several steps. Firstly, the NdeI site in the vector part of pAIA50 was eliminated by linearization with NdeI, filling in of ends and religation. This yielded pAIA55. Subsequently, two mutant fragments were derived from bphA1-LB400 by PCR, using primer pairs LP11/LP16 or LP13/LP18. The first one contained the wild-type (WT) MluI site and an NdeI site at positions 24052410 of the LB400 bph locus (Erickson & Mondello, 1992
), introduced by silent mutations within the 3'-terminal PCR primer. The second mutant fragment contained the WT AgeI site and an XhoI site at positions 24692474 of the LB400 bph locus, introduced by silent mutations within the 5'-terminal PCR primer. These two overlapping PCR fragments were agarose gel-purified and fused by OE-PCR with primers LP11 and LP18. The resulting mutant bphA1-LB400 fragment containing NdeI and XhoI sites was used to replace the WT MluIAgeI fragment of pAIA55 to yield pAIA6000. Replacement of its MluINdeI fragment by the respective fragment from bphA1-P6, obtained by PCR with primers LP1 and LP14, yielded bphA1-H09 and pAIA509, respectively. The bphBC genes of pAIA6000, -503, -504 and -509, respectively, were deleted by cleavage with BspEI and recircularization to yield pAIA100, -103, -104 and -109, respectively. Hybrids bphA1-H10, -H20 and -H30 were constructed by modifications of pAIA100. To obtain bphA1-H10, the AgeIAscI fragment of pAIA100 was exchanged for the respective fragment of pAIA504, which was PCR-synthesized using primers LP15 and LP10. The upstream primer introduced an AgeI site. This yielded pAIA110. To generate bphA1-H20, firstly the required region of pAIA504 was amplified using a hybrid LB400/P6 forward primer (LP19) and a reverse primer annealing at the AscI site (LP10). This segment was fused by OE-PCR with the respective upstream segment from pAIA100 which was synthesized with primers LP17 and LP20. The derived XhoIAscI fragment was used to replace the respective fragment of pAIA100, yielding pAIA120. To construct bphA1-H30, the required P6 region was amplified from pDM10 using primers LP23 and LP24. This product was fused by OE-PCR with up- and downstream pAIA100 segments which had been amplified with primers LP21 and LP22 or LP25 and LP26, respectively. The fusion product was cleaved with MluI and NdeI, and used to replace the respective fragment of pAIA100 to yield pAIA130. The correct sequence of all cloned PCR-synthesized fragments was verified by sequencing.
DNA sequencing.
This was carried out as described by Hofer et al. (1994).
Visualization of bphA1 gene products.
Resting cells of E. coli BL21(DE3)[pLysS] harbouring pAIA6000 or derivatives (see below) were concentrated 10-fold and lysed by two different methods. For SDS lysis, 2·5 µl cells were mixed with an equal volume of 2x cracking buffer (Tabor & Richardson, 1985 ) and heated to 95 °C for 2 min prior to gel electrophoresis. Alternatively, concentrated resting cells were disrupted by two passages through a French press (Aminco) at 138 MPa. A portion (2·5 µl) of the homogenate was mixed with 1 vol. 2x cracking buffer and heated as above. Electrophoresis was carried out in 0·1% SDS/12% polyacrylamide gels as described previously (Hofer et al., 1993
). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al., 1989
).
Measurement of in vivo BDO activity.
With cells harbouring plasmids containing bphA1A2A3A4BC, BDO activity was assessed on plates by adding excess solid biphenyl to the lids and monitoring formation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate by eye. For quantification, resting cells of E. coli BL21(DE3)[pLysS] harbouring pAIA6000 or derivatives (see below) at a concentration of 0·2 OD600 units were incubated with biphenyl (final concentration 0·25 mM) with shaking (175 r.p.m.) at 30 °C, and the formation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate was monitored photometrically (A434). With plasmids harbouring bphA1A2A3A4, activity was assayed likewise, with the exception that the resting cells (final concentration 0·1 OD600 unit) were mixed with resting cells harbouring pDD372 (final concentration 0·2 OD600 units), which directs the syntheses of 2,3-dihydro-2,3-dihydroxybiphenyl-2,3 dehydrogenase and 2,3-dihydroxybiphenyl-1,2 dioxygenase.
Preparation of resting cells.
E. coli BL21(DE3)[pLysS] harbouring pAIA6000 or derivatives was grown at 37 °C in LB medium with antibiotics to an OD600 between 1·1 and 1·3. IPTG was added to 0·4 mM final concentration and the incubation continued at 30 °C for another 30 min. Cells were harvested, washed with 50 mM sodium phosphate buffer, pH 8·0, containing 5 mM glucose and resuspended in the same buffer to a final concentration of OD600=5·0. As WT dioxygenase activity from strain P6 could not be measured in E. coli (McKay et al., 1997 ; Chebrou et al., 1999
), the respective genes were expressed in P. putida KT2442 as previously described (McKay et al., 1997
). Resting cells of P. putida were prepared as above.
Resting cell assays.
Single chlorobiphenyls (CBs) were added to 1 ml resting cells to a final concentration of 0·75 mM. The Teflon-sealed tubes were shaken at 175 r.p.m. at 30 °C for 24 h. Thereafter, 50 µl 25 mM 2,2'-dihydroxybiphenyl was added as a standard and the suspensions were extracted with 1 vol. ethylacetate by inverting several times. A portion (10 µl) of the organic phase was analysed in a high performance liquid chromatograph (Shimadzu LC10AD) equipped with a diode array detector and an SC Lichrosphere 100 RP8 5 µm column (length, 125 mm; internal diameter, 4·6 mm) (Bischoff). The aqueous eluent contained 1 ml 85% ortho-phosphoric acid and 600 ml methanol l-1. The flow rate was 1 ml min-1. Metabolites were quantified from HPLC peak areas which had been normalized to the internal standard. Percentages of metabolites were calculated on the basis that the sum of all metabolites formed by a given enzyme represents 100%. HPLC peak areas were derived from absorptions at the wavelengths indicated in Table 3. As molar extinction coefficients are unknown, molar amounts could not be determined. Thus, the given percentages of metabolites do not necessarily represent molar ratios.
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RESULTS |
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The distributions of products obtained with WT and chimaeric enzymes are shown in Fig. 5. Analytical data of the metabolites are given in Table 3
. In Fig. 5
, the columns representing marker metabolites that are particularly prominent with BDO-LB400 or -P6 are shown hatched or solid, respectively. Correlation with Table 3
shows that the four marker metabolites for BDO-LB400 reflect dehalogenating attacks at carbons 2 and 3 of 2,2'-, 2,3'- or 2,4'-CB, respectively, and the meta,para-dioxygenation of 2,5,2',5'-CB. The three marker metabolites for BDO-P6 were formed by non-dechlorinating ortho,meta-dioxygenation of the meta-chlorinated ring of 2,3'- and 3,4'-CB, and of the para-chlorinated ring of 2,4'-CB.
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DISCUSSION |
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The hybrid dioxygenase BDO-H04 possessed substrate and product spectra that closely resembled those of the enzyme from R. globerulus P6. This result, in view of the 43% sequence difference between BphA2-LB400 and -P6, suggests that the ß subunits exert no major influence on the structure of the active site. We note that these findings contrast those of Hurtubise et al. (1998) and Chebrou et al. (1999)
, who reported a significant contribution of the ß subunit to BDOsubstrate interaction. However, a recent publication from this group (Barriault et al., 2001
) reported no major influence of the small subunit on substrate specificity. Based on all fusions generated, the
subunit may be divided into eight segments, designated A to H (Fig. 2
). The N-terminal region
A harbours the Rieske iron-sulphur cluster (Fig. 2
). In BDO-H04,
A was derived from BDO-LB400. The corresponding region of BDO-P6 differs in 70 (42%) of the residues. Thus, the highly similar regiospecificity of dioxygenation by BDO-H04 and BDO-P6 makes it unlikely that region
A significantly participates in formation of the substrate-binding pocket, although an
subunit sequence alignment with a naphthalene dioxygenase, for which three-dimensional crystal structures have been determined (Kauppi et al., 1998
; Carredano et al., 2000
), suggests that the N-terminal residues up to about position 55 are part of the catalytic domain (Fig. 2
) that harbours the active site. Barriault et al. (2001)
exchanged the
A regions between the BDOs from Comamonas testosteroni B-356 and strain LB400, which differ at 28 positions. Whilst the B-356 variant containing
A from strain LB400 behaved very similarly to the B-356 WT, the reverse replacement showed significant differences in turnover of two of the four CBs assayed. This suggests a context effect on these exchanges (see below).
Chimaeras BDO-H10 and -H20 examined the other end of the subunit. Their product profiles were almost identical to that of BDO-LB400. The exchanged regions at the C terminus differ by almost 40% from the LB400 sequence. This indicates that the C-terminal 75 aa (regions
G and
H) exert an almost negligible effect on substrate dioxygenation.
Apparently, in BDO-H03, -H09 and -H30, the structure of the active site was influenced by peptide segments from both sources. The conversion of 2,4'-CB indicated a significant difference between BDO-H03 and -H04. As regions G and
H have been shown to be of minor importance, this result highlights a significant effect of region
F. Previously, Kimura et al. (1997)
and Mondello et al. (1997)
identified a major contribution of aa 377 (LB400 numbering) on substrate specificity. This is one of the three amino acid differences between regions
F of the LB400 and P6 subunits.
Differences in CB dioxygenation between BDO-H09 and -H03 clearly demonstrate the importance of region E, in agreement with findings made in the LB400/KF707 system (Erickson & Mondello, 1993
; Kimura et al., 1997
; Mondello et al., 1997
). However, the difference in CB dioxygenation between BDO-H09 and BDO-LB400 was not expected on the basis of those studies. It revealed that sequence elements N-terminal to position 320 significantly influence the substrate-binding site. A first indication for this was provided by Kumamaru et al. (1998)
who found that a random BDO variant which had obtained substitutions of aa 255, 258 and 303 showed an altered substrate specificity. The segment exchanged in BDO-H09, by analogy with other ARHDOs, is likely to comprise amino acid ligands of the active site mononuclear iron (Fig. 2
) (Jiang et al., 1996
; Butler & Mason, 1997
; Kauppi et al., 1998
). We therefore constructed a hybrid, BDO-H30, in which primarily these sequence elements were replaced. Also this variant clearly behaved differently from BDO-LB400, demonstrating that exchange of this subregion indeed altered enzymesubstrate interactions. Although investigations of different ARHDOs are of limited comparability, we note that Beil et al. (1998)
and Parales et al. (2000)
reported effects of amino acid substitutions at five or two positions, respectively, within the corresponding regions of a toluene or a naphthalene dioxygenase, respectively. Barriault et al. (2001)
, however, exchanging a region between BDOs of strains LB400 and B-356 that comprises the region substituted in BDO-H30, found only a minor influence on substrate specificity. These differences in results may be a consequence of the different assays used. They may also be due to the fact that amino acid replacements in these experiments (partly) differ in positions, in types of substitutions and in flanking sequences (context dependence). If only a fraction of the amino acid residues that determine a given property are exchanged, context dependence of the effects of such replacements is inevitable. This and other publications (Beil et al., 1998
; Kauppi et al., 1998
; Carredano et al., 2000
; Parales et al., 2000
) suggest that the structure of the substrate-binding site is determined by segments that are rather distant in the sequence. Thus, context dependence is expected for the effects of individual exchanges of such segments on the structure of the binding site. Therefore, generalizations in the interpretation of experiments must be made with caution.
Problems were encountered in the biosynthesis of the BDO from R. globerulus P6 in recipient organisms. Whilst active BDO-P6 was obtained in P. putida cells grown under specific conditions, neither activity nor BDO proteins were detected in E. coli (McKay et al., 1997 ). Numerous examples indicate that insufficient initiation of translation is a major obstacle in the expression of cloned genes. Inefficient initiation in most cases is related to the new combination of 5'-untranslated and/or -translated gene regions. Recently, Chebrou et al. (1999)
successfully used a different expression vector additionally encoding a 5'-terminal His-tag to achieve synthesis of the BDO
and ß subunits from strain P6 in E. coli. However, these authors encountered another problem. The hydroxylase formed was catalytically inactive due to incorrect assembly of the iron-sulphur cluster. Results of the present study indicate that both difficulties may be circumvented by one and the same approach. The generation of P6-like BDO activity in E. coli by a hybrid enzyme exemplifies that fusion of the problematic gene with a helper cistron is a feasible strategy to obtain a specific ARHDO activity in a host organism that is inappropriate to carry out synthesis of the respective WT enzyme in an active form.
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ACKNOWLEDGEMENTS |
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Received 31 December 2001;
revised 29 April 2002;
accepted 7 May 2002.