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Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
Correspondence
Andreas Stolz
Andreas.Stolz{at}imb.Uni-Stuttgart.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-ketoadipate pathway via 4-sulfocatechol and 3-sulfomuconate. It was previously proposed that the further metabolism of 3-sulfomuconate is catalysed by modified 3-carboxy-cis,cis-muconate-lactonizing enzymes (CMLEs) and that these ‘type 2’ enzymes were different from the conventional CMLEs (‘type 1’) from the protocatechuate pathway in their ability to convert 3-sulfomuconate in addition to 3-carboxy-cis,cis-muconate. In the present study the genes for two CMLEs (pcaB2S1 and pcaB2S2) were cloned from H. intermedia S1 and A. radiobacter S2, respectively. In both strains, these genes were located close to the previously identified genes encoding the 4-sulfocatechol-converting enzymes. The gene products of pcaB2S1 and pcaB2S2 were therefore tentatively identified as type 2 enzymes involved in the metabolism of 3-sulfomuconate. The genes were functionally expressed and the gene products were shown to convert 3-carboxy-cis,cis-muconate and 3-sulfomuconate. 4-Carboxymethylene-4-sulfo-but-2-en-olide (4-sulfomuconolactone) was identified by HPLC-MS as the product, which was enzymically formed from 3-sulfomuconate. His-tagged variants of both CMLEs were purified and compared with the CMLE from the protocatechuate pathway of Pseudomonas putida PRS2000 for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-cis,cis-muconate. Also the CMLE from P. putida converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-cis,cis-muconate as substrate. Thus it was demonstrated that in the 4-sulfocatechol pathway, distinct CMLEs are formed, which are specifically adapted for the preferred conversion of sulfonated substrates.
The GenBank/EMBL/DDBJ accession numbers for the pcaB2S2 and pcaB2S1 sequences reported in this paper are AY769867 and AY769868, respectively.
Present address: Institut Pasteur, 25 Rue du Dr Roux, F-75015 Paris, France.
Present address: Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, D-70736 Fellbach, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Laboratory studies have shown that a sulfonic acid substituent usually significantly decreases the rates of biodegradation, and several examples of the accumulation of sulfonated aromatics in the environment have been described (Alexander & Lustigman, 1966; Alonso et al., 1999; Knepper, 2002; Riediker et al., 2000; Ruckstuhl et al., 2002; Wellens, 1990). The microbial degradation of aromatic sulfonic acids has mainly been studied using simple benzenesulfonates and naphthalenesulfonates as model compounds. These studies demonstrated that sulfonated substrates are in most cases initially desulfonated by the action of ring-hydroxylating dioxygenases to the corresponding diols (Brilon et al., 1981; Cook et al., 1999; Nörtemann et al., 1986; Ohe et al., 1990; Thurnheer et al., 1990; Wittich et al., 1988). In contrast, it has been found that 4-aminobenzenesulfonate (sulfanilate) was initially deaminated by a co-culture of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2 to 4-sulfocatechol (Feigel & Knackmuss, 1988). More recently, 4-sulfocatechol has also been described as an intermediate formed during the degradation of 1,3-benzenedisulfonate and linear alkylbenzenesulfonates (Contzen et al., 1996; Schulz et al., 2000; Dong et al., 2004; Schleheck et al., 2004). 4-Sulfocatechol thus appears to be a central intermediate for the degradation of substituted sulfonated benzenes, which are released by humans in enormous quantities (>106 tonnes annually) into the environment.
It was demonstrated for the sulfanilate-degrading mixed culture that 4-sulfocatechol was oxidized to 3-sulfomuconate by specifically adapted forms of protocatechuate 3,4-dioxygenases. These ‘type 2’ enzymes were therefore distinguished from the classical protocatechuate 3,4-dioxygenases (‘type 1’) which convert only protocatechuate (Feigel & Knackmuss, 1993; Hammer et al., 1996). The genes encoding type 2 protocatechuate 3,4-dioxygenases (P34OIIs) have been cloned from H. intermedia S1 and A. radiobacter S2, and significant sequence similarities have been found among the genes encoding the type 1 and type 2 enzymes (Contzen & Stolz, 2000; Contzen et al., 2001).
It has been suggested that 3-sulfomuconate is further converted in a cycloisomerization reaction to a sulfonated lactone (Fig. 1). This reaction is analogous to the cycloisomerization of 3-carboxy-cis,cis-muconate to 4-carboxymuconolactone found in the protocatechuate branch of the -ketoadipate pathway, which is catalysed by 3-carboxy-cis,cis-muconate-lactonizing enzymes (CMLEs). The CMLE from Pseudomonas putida has been purified and the stereochemistry of the turnover of 3-carboxy-cis,cis-muconate has been analysed in some detail. In addition, the gene has been cloned and sequenced, and the crystal structure of the enzyme has been determined (Ornston, 1966; Williams et al., 1992; Yang et al., 2004).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Feigel & Knackmuss, 1988, 1993). The strains were routinely cultivated separately in SHPG-medium as described elsewhere (Contzen et al., 2000; Feigel & Knackmuss, 1988, 1993).
Pseudomonas putida PRS2000 (Hughes et al., 1988) was kindly provided by J. Gröning and M. Schlömann (TU Freiberg, Germany). The organism was grown at 30 °C on a rotary shaker (100 r.p.m.) in a mineral medium according to Dorn et al. (1974) with protocatechuate (7.5 mM) as sole source of carbon and energy. For isolation of genomic DNA, the strain was cultivated in liquid culture with NB [8 g dehydrated Nutrient Broth powder (Difco) and 5 g NaCl l–1].
Escherichia coli DH5
, JM 109, BL21(DE3)(pLysS) and BL21(DE3)(pLysS) Star (Invitrogen) were used as host strains for recombinant DNA work. E. coli strains were routinely cultured in Luria–Bertani (LB) medium supplied with ampicillin (100 µg ml–1) or kanamycin (50 µg ml–1), if appropriate.
E. coli BL21(DE3)(pLysS)(pETS2-X-II) (Contzen & Stolz, 2000) was used for the synthesis of 3-carboxy-cis,cis-muconate and 3-sulfomuconate from protocatechuate and 4-sulfocatechol, respectively.
Plasmids and DNA manipulation techniques.
Plasmid pBluescript II SK(+) was used for standard cloning experiments (Alting-Mees et al., 1992). The plasmid vectors pJOE3075 and pAC28 were used for high levels of gene expression (Kholod & Mustelin, 2001; Stumpp et al., 2000). The characteristics of all plasmids used are given in Table 1.
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. The genomic DNA from A. radiobacter S2 was extracted using a DNeasy Tissue Kit (Qiagen) and the genomic DNA of P. putida using an E.Z.N.A Bacterial DNA Kit (Peqlab Biotechnologie). Plasmid DNA from E. coli DH5 was isolated with a GFX Micro Plasmid Prep kit (Pharmacia). Digestion of DNA with restriction endonucleases (MBI Fermentas), electrophoresis and ligation with T4 DNA ligase (MBI Fermentas) were performed according to standard procedures (Sambrook et al., 1989). Transformation of E. coli was done by the method of Chung et al. (1989). For cloning of certain PCR products a T-vector was prepared as described by Marchuk et al. (1991).
PCR.
Oligonucleotides were custom-synthesized (Eurogentec) according to known or deduced sequences from various CMLEs (Table 2). PCR mixtures (50 µl) for the amplification of genomic DNA contained 50 pmol each primer, 0.1–0.2 µg genomic DNA, 0.2 mM each deoxynucleotide triphosphate, Taq DNA polymerase (2–2.5 U) and the corresponding reaction buffer (Eppendorf).
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). The template was prepared by digesting chromosomal DNA (200 ng) of strain S1 for 1 h at 37 °C with PauI and religating the fragments obtained with T4 DNA ligase. The primers for this PCR (pcaBS1_1930F and pcaBS1_1888R; Table 2
) were deduced from the fragment of pcaB2S1 obtained previously. The following PCR program was used: an initial denaturation (95 °C, 1 min) was followed by 30 cycles consisting of an annealing step (60 °C, 1 min), a polymerization step (72 °C, 4 min) and a denaturation step (95 °C, 1 min). This resulted in the amplification of an approximately 2.4 kb fragment.
Construction of expression plasmids for the production of the CMLEs from A. radiobacter S2 (ArCMLE2), H. intermedia S1 (HiCMLE2) and P. putida PRS2000 (PpCMLE1) in E. coli.
The genes pcaB2S1, pcaB2S2 and pcaB encoding HiCMLE2, ArCMLE2 and PpCMLE1, respectively, were amplified by PCR. The primers used simultaneously introduced NdeI sites upstream and BamHI sites downstream of the genes (see Table 2). The PCR products were then cleaved with NdeI and BamHI and inserted into NdeI/BamHI-cut expression vectors, resulting in amino-terminally His-tagged enzyme variants.
The pcaB2S2 gene from A. radiobacter S2 was amplified by PCR using the oligonucleotide primers CMLEII-X-N and CMLEII-X-C. The following PCR program was used: an initial denaturation (94 °C, 1 min) was followed by 30 cycles consisting of an annealing step (65 °C, 1 min), a polymerization step (72 °C, 2 min) and a denaturation step (94 °C, 1 min). The amplified and digested DNA fragment was subsequently cloned into the expression vector pAC28 (Kholod & Mustelin, 2001).
HiCMLE2 was amplified using Pfu DNA polymerase (Stratagene) from the genomic DNA of H. intermedia S1 using primers pcaB2S1_X_N and pcaB2S1_X_B (Table 2), which were derived from the DNA sequence of the 2.4 kb DNA fragment obtained by inverse PCR (see above). The following PCR program was used: an initial denaturation (96 °C, 2 min) was followed by 30 cycles consisting of an annealing step (63.5 °C, 1 min), a polymerization step (72 °C, 2 min) and a denaturation step (96 °C, 30 s). The amplified product was cleaved with NdeI and BamHI and ligated into pJOE3075 (Stumpp et al., 2000). Finally, E. coli JM 109 was transformed with the resulting plasmid pSBCMC2S1. A variant of the enzyme with an amino-terminal His-tag was constructed by cutting out pcaB2S1 from pSBCMC2S1 using NdeI and BamHI and cloning it into the expression vector pAC28, giving plasmid pSHCMC2S1.
The pcaB gene was amplified using primers pPcaB-X-N2 and pPcaB-X-C2 (Table 2), which were deduced from the known nucleotide sequence of the gene [National Center for Biotechnology Information (NCBI) no. L17082]. The following PCR program was used: an initial denaturation (94 °C, 2 min) was followed by 30 cycles consisting of an annealing step (65 °C, 30 s), a polymerization step (72 °C, 1 min) and a denaturation step (94 °C, 30 s). The amplified product was cleaved with NdeI and BamHI and cloned into pAC28, giving pSHPpCMLE.
Expression of HiCMLE2, ArCMLE2 and PpCMLE1 in E. coli.
The three CMLEs were heterologously produced as amino-terminally His-tagged enzyme variants using E. coli BL21(DE3)(pLysS) Star(pSHCMC2S2) for ArCMLE2, E. coli BL21(DE3)(pLysS) Star(pSHCMC2S1) for HiCMLE2 and E. coli BL21(DE3)(pLysS) Star(pSHPpCMLE) for PpCMLE. The strains were grown in 300 ml LB medium (plus 50 µg kanamycin ml–1) and the expression of the CMLEs was induced with 1 mM IPTG. The cells were harvested by centrifugation, resuspended in Tris/HCl buffer (50 mM, pH 8.0), disintegrated using a French Press, and cell extracts were prepared by centrifugation at 100 000 g for 30 min at 4 °C. The CMLEs were purified using an Ni-NTA-column (25 ml; Qiagen) attached to an FPLC-apparatus (Pharmacia). The column was equilibrated with Tris/HCl (50 mM, pH 8.0), NaCl (300 mM), imidazole (20 mM) and DTT (1 mM). The CMLEs were eluted from the column by increasing the imidazole concentration to 200 mM in the buffer system described above. Fractions (5 ml each) were collected, the fractions with the CMLEs (usually fractions 3–5) were pooled and the elution buffer was removed using an ultrafiltration unit (20 ml Concentrator, 100 000 molecular mass cut-off; Vivascience). Finally, the concentrated enzyme solution was diluted with Tris/HCl (50 mM, pH 8.0). This procedure resulted in enzyme preparations of HiCMLE2 and ArCMLE2 which appeared on silver-stained gels to be greater than 99 % pure for both enzymes.
Nucleotide sequence analysis.
The DNA sequences were determined by dideoxy-chain termination with double-stranded DNA of overlapping subclones in an automated DNA sequencing system (ALF-Sequencer; Amersham-Pharmacia) with fluorescently labelled primers.
Sequence analysis, database searches and comparisons were done with the Lasergene software package, version 5 (DNASTAR) and the BLAST search facility at NCBI (Altschul et al., 1997). The alignments of the CMLEs were obtained with the program CLUSTALX using default parameters.
Estimation of protein concentration and enzyme assays.
The protein content of cell-free extracts was determined by the method of Bradford (1976). Bovine serum albumin was used as standard. One unit of enzyme activity was defined as the amount of enzyme that converts 1 µmol substrate min–1.
CMLE activity was measured using the spectrophotometric assay described by Ornston & Stanier (1966). The assays contained 100 µM 3-carboxy-cis,cis-muconate (synthesized enzymically from protocatechuate, see below) and 50 mM Tris/HCl buffer (pH 8.0) in a final volume of 1 ml. The decrease of absorption was determined at 260 nm. The reaction rates were calculated by using a molar extinction coefficient of 
260=7.3 mM–1 cm–1.
The turnover of 3-sulfomuconate was analysed by HPLC. The reaction mixtures contained 50 mM Tris/HCl buffer (pH 8.0) and 100 µM 3-sulfomuconate (synthesized enzymically from 4-sulfocatechol; see below). The reactions were usually monitored for 14 min, every 2 min aliquots (60 µl each) were removed, and the reactions were terminated in liquid nitrogen. The samples were separately thawed immediately before the HPLC analysis and the reaction rates were calculated from the time-dependent decrease in 3-sulfomuconate concentration.
Determination of molecular mass.
The relative molecular masses of the native enzymes were determined by gel filtration using a Superdex 200 prep grade column (Amersham Biosciences) calibrated with an HMW Gel Filtration Calibration Kit (Amersham Biosciences). The subunit sizes were determined by SDS-PAGE with a Premixed Protein Molecular Mass Marker 14.4–97.4 kDa Kit (Roche) as reference proteins.
SDS-PAGE.
SDS-PAGE was performed by the method of Laemmli (1970) and the gels were routinely stained with Coomassie blue. In some experiments the gels were silver-stained using a Dodeca Silver Stain Kit (Bio-Rad).
HPLC.
The turnover of 4-sulfocatechol and 3-sulfomuconate was analysed by reversed-phase HPLC (pump model 510 equipped with a photodiode array detector, model 996, and Millenium Chromatography Manager 2.0; Waters Associates). A reversed-phase column (250x4.0 mm i.d., packed with 3 µm particles of Nucleosil C18) was used. The mean flow rate was 1 ml min–1. The separated compounds were detected photometrically at 210 nm using a photodiode array detector. The solvent system consisted of 98.9 % (v/v) water, 1 % (v/v) methanol and 0.1 % (v/v) H3PO4. The mean retention times of 4-sulfocatechol, 3-sulfomuconate and 4-sulfomuconolactone under these chromatographic conditions were 3.7, 3.4 and 4.3 min, respectively.
Liquid chromatography (LC)-MS.
Products were identified by using LC-MS (HP1100; Agilent) coupled to a triple quadrupole mass spectrometer (Quattro LC; Micromass) using electrospray ionization in the negative ion mode. Substrate solutions before and immediately after addition of enzyme and 20 min after addition of the enzyme were injected (20 µl) into the HPLC system without any pretreatment. Analytes were separated by ion-pair chromatography on a Luna C18(2) 3 µm column, 15 cmx3 mm i.d. at 40 °C. Eluent A was H2O/MeOH (80 : 20, v/v) and eluent B, H2O/MeOH (5 : 95 v/v) with 5 mM tributylamine and 5 mM acetic acid. The gradient was 22 % (v/v) B at 0 min, 22 % B at 2 min, 90 % B at 10 min, 90 % B at 14 min and 22 % B at 15 min, ready for injection after 22 min. A diode array detector and the mass spectrometer were coupled in series. The mass spectrometric interface was operated at a cone voltage of 18 V and a capillary voltage of 2.9 kV. The probe temperature was 220 °C and the source block temperature 120 °C. Product ion spectra were recorded at collision energies of 10 eV and 15 eV with a scan rate of 0.5 s.
Preparation of 3-carboxy-cis,cis-muconate and 3-sulfomuconate.
The substituted muconates were prepared enzymically using protocatechuate dioxygenase activity (type 2), according to a method described by Ornston & Stanier (1966) for the preparation of 3-carboxy-cis,cis-muconate. The source of the required dioxygenase activity was E. coli BL21(DE3)(pLysS)(pETS2-X-II), which heterologously expresses P34OII from A. radiobacter S2 under the control of a phage T7 promoter (Contzen & Stolz, 2000). The strain was grown in 300 ml LB medium (plus 100 µg ampicillin ml–1) and the expression of P34OII was induced with IPTG (0.4 mM). The cells were harvested by centrifugation, resuspended in Tris/HCl buffer (50 mM, pH 8.0), disintegrated and cell extracts prepared. The cell extracts (about 2 ml with a protein content of about 40 mg ml–1) were mixed with 10 ml Tris/HCl (50 mM, pH 8.0) and protocatechuate or 4-sulfocatechol was added (4 mM each). The reaction mixtures were incubated at 30 °C on a laboratory shaker (100 r.p.m.).
The cell-free extract incubated with 4-sulfocatechol was analysed simultaneously by overlay spectra and HPLC analysis. The overlay spectra demonstrated the characteristic decrease in absorbance at 280 nm due to the turnover of 4-sulfocatechol (Hammer et al., 1996). HPLC analysis with a reverse phase column [solvent system 98.9 % (v/v) water, 1 % (v/v) methanol, 0.1 % (v/v) H3PO4] indicated that 4-sulfocatechol (Rt=3.7 min; in situ-recorded 
max=232 nm, 281 nm) was converted to a new metabolite (Rt=3.5 min; max=205 nm). The turnover of 4-sulfocatechol to 3-sulfomuconate was additionally verified by HPLC-MS/MS analysis. A new signal occurred (Rt=9.9 min) with a molecular anion mass [M-H]– of m/z 221 that corresponds to sulfomuconate (C6H6O7S). Fragment ions detected in product ion spectra indicated the presence of one sulfonate moiety and two carboxylate groups: m/z 177 (M-H-CO2), 149 (177-CO), 139 (M-H-H2SO3), 113 (177-CO2), 95 (139-CO2) and 81 (
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The solutions of the substituted muconates were separated from the proteins by ultrafiltration (30 000 molecular mass cut-off; Vivaspin). The resulting filtrate containing 3-carboxy- or 3-sulfomuconate was used for the enzymic tests.
Chemicals.
The chemicals used were obtained from Aldrich, Fluka, Merck and Sigma. 4-Sulfocatechol was synthesized according to the procedure described by Quilico (1927).
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). In the course of the present study, the DNA insert on pMCS2-2 was completely sequenced and it was found that this gene, designated pcaB2S2, encodes ArCMLE2, a protein of 407 aa with 37–42 % sequence identity to previously described CMLEs (Fig. 2).
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). Various attempts to obtain DNA fragments adjacent to pcaH2G2 by partial inverse PCR only resulted in the cloning of a 1180 bp fragment which contained a putative transposase gene about 270 bp downstream of pcaH2G2. Therefore, sequence alignments were performed using the gene sequence obtained from strain S2 (see above) and those of the CMLEs from Streptomyces sp. strain 2065 (NCBI no. AAD40814), Streptomyces coelicolor (T35016), Pseudomonas aeruginosa PAO1 (NP_248921), P. putida (P32427), Bradyrhizobium japonicum USDA110 (O31385), Acinetobacter sp. ADP1 (Q59092) and Agrobacterium tumefaciens C58 (AAK88905). These alignments demonstrated the presence of two highly conserved regions among all sequences, and the sequences (encoding aa 37–45 and 280–288 in ArCMLE2) were used to design PCR primers (Table 2). A PCR using these primers and genomic DNA from strain S1 resulted in the amplification of a DNA fragment with the expected size (about 750 bp). The fragment was sequenced and shown to encode part of a putative CMLE. Because in A. radiobacter S2 pcaH2G2 and pcaB2S2 are physically connected, another PCR experiment was performed with genomic DNA from strain H. intermedia S1 using one of the primers deduced from the conserved regions in the CMLEs (c-S1-as/a; Table 2) and another one (S1-pcaG/se; Table 2) from the region downstream of pcaG2 known from previous work. This PCR resulted in the amplification of an approximately 2 kb DNA fragment. Sequencing of this fragment demonstrated that the putative pcaB2S1 was situated downstream of the putative transposase which, in turn, was located downstream of pcaH2G2. The missing part of pcaB2S1 was finally obtained using partial inverse PCR (see Methods). The gene encoded HiCMLE2, a protein of 453 aa (HiCMLE2) with significant homology to known CMLEs over its complete length (Fig. 2).
Comparison of the amino acid sequences of ArCMLE2 and HiCMLE2 with other CMLEs from different bacteria
A comparison of the deduced amino acid sequences of HiCMLE2 and ArCMLE2 and the sequences of other proven or putative CMLEs from the NCBI database clearly demonstrated that the two type 2 enzymes were more closely related to each other than to the other CMLEs (Fig. 3). The two presumed type 2 enzymes showed 50 % sequence identity, which was significantly higher than the sequence identities of 33–43 % observed between the two type 2 enzymes and all other previously described CMLEs (including all sequences of presumed CMLEs from various genome sequencing projects).
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). This indicates that no strictly conserved 4-sulfocatechol degradation operon exists and that the two pathways were not established in both strains by recent gene exchange events.
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), giving plasmids pSHCMC2S1 and pSHCMC2S2, respectively. This resulted in the formation of carboxy- and amino-terminally His-tagged proteins. The expression of the genes by the addition of IPTG resulted in the formation of additional peptides in the crude extracts. The sizes of the additional bands were estimated by SDS-PAGE for both HiCMLE2 and ArCMLE2 at approximately 42 kDa. The CMLE activities of the recombinant E. coli strains were tested using the spectrophotometric enzyme assay described by Ornston & Stanier (1966). Thus it was shown that the cell extracts from E. coli BL21(DE3)(pLysS)(pSHCMC2S1) and E. coli BL21(DE3)(pLysS)(pSHCMC2S2) converted 3-carboxy-cis,cis-muconate with specific activities of 0.67 and 0.5 U (mg protein)–1, respectively.
Turnover of 3-sulfomuconate by HiCMLE2 and ArCMLE2
Cell extracts were prepared from E. coli BL21(DE3)(pLysS) Star carrying plasmids pSHCMC2S1 or pSHCMC2S2 and incubated with 3-sulfomuconate (1–4 mM). These reactions were analysed by HPLC because 3-sulfomuconate, unlike most other substituted muconates, does not have pronounced absorbance at 260 nm (Feigel & Knackmuss, 1993). HPLC analysis with a reverse phase column (using the same solvent system as above) demonstrated that 3-sulfomuconate (Rt=3.5 min; max=205 nm) was converted to a new metabolite (Rt=4.2 min; max=215 nm). The subsequent analysis of the reaction by HPLC-MS/MS confirmed the turnover of 3-sulfomuconate to 4-carboxymethylene-4-sulfo-but-2-en-olide (4-sulfomuconolactone). A new signal was detected (Rt=9.0 min) with an m/z of 221 for the molecular anion [M-H]–. Less fragmentation occurred than for sulfomuconate, as the cyclic lactone system remained intact and only the exocyclic functional groups were split off as fragments: 177 (M-H-CO2), 139 (M-H-H2SO3), 95 (139-CO2) and 81 (
). No reactions were observed in control experiments without added cell extracts or with cell extracts from E. coli which did not express the recombinant enzymes.
The recombinant CMLEs were purified by affinity chromatography using their His-tags. The purified enzymes showed (determined with 3-carboxy-cis,cis-muconate as substrate) pH optima in Tris/HCl buffer at pH 8.0 and about 50 % of their maximal activity in Tris/HCl, pH 9.0, or histidine/HCl, pH 5.8. The enzymes showed significantly reduced activities in sodium/potassium phosphate/citrate buffers compared to histidine/HCl buffers at the same pH, as found previously for the CMLE from P. putida (Ornston, 1966).
In the spectrophotometric tests with 3-carboxy-cis,cis-muconate, vmax values of 1.5 and 1.3 U (mg protein)–1 were determined for HiCMLE2 and ArCMLE2, respectively (Table 3). These values were significantly lower than those described for the CMLE from P. putida [547 U (mg protein)–1] (Ornston, 1966). Furthermore, HiCMLE2 and ArCMLE2 also showed slightly higher Km values for 3-carboxy-cis,cis-muconate than the enzyme from P. putida (Table 3).
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Turnover of 3-sulfomuconate by PpCMLE from P. putida PRS2000
We tested if also the ‘archetypal’ CMLE from the protocatechuate pathway of P. putida PRS2000(PpCMLE1) could convert 3-sulfomuconate. Cell extracts were prepared from cells of P. putida PRS2000 grown with protocatechuate (to induce the formation of PpCMLE1). The cell extracts converted 3-carboxy-cis,cis-muconate and surprisingly also 3-sulfomuconate with specific activities of 2.0 and 0.13 U (mg protein)–1, respectively. To confirm the ability of PpCMLE1 to convert 3-sulfomuconate, the gene was amplified from the chromosomal DNA of P. putida PRS2000 and cloned into the expression vector pAC28 (see Methods). The enzyme was expressed as a His-tagged enzyme variant and partially purified on an Ni-NTA column to a specific activity of 240 U mg–1 with 3-carboxy-cis,cis-muconate as substrate [compared to a vmax reported for the purified enzyme of 457 U (mg protein)–1 by Ornston (1966)]. The same enzyme preparation was incubated with 3-sulfomuconate (0.5, 1, 2 and 3 mM) in the HPLC assay used for the CMLEs. Thus it was found that PpCMLE indeed converted 3-sulfomuconate. In these tests the highest reaction rates were observed with 0.5 mM 3-sulfomuconate [8.5 U (mg protein)–1] and a pronounced decrease in the reaction rates was observed using higher substrate concentrations.
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DISCUSSION |
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-ketoadipate pathway. This became evident from the range of substrates converted by both groups of enzymes and the sequence data obtained. It was found previously that the P34OIIs from H. intermedia S1 and A. radiobacter S2 converted 4-sulfocatechol and protocatechuate (Contzen et al., 2001). In accordance with this observation, the present study demonstrated that ArCMLE2 and HiCMLE2 converted carboxylated and sulfonated muconates. Furthermore, it was previously shown by multiple sequence alignments that both subunits of the P34OIIs from strains S1 and S2 clustered together with the corresponding subunits of the protocatechuate 3,4-dioxygenases from various protocatechuate-degrading organisms (Contzen & Stolz, 2000; Contzen et al., 2001) and here we show that the two type 2 CMLEs are also homologues of the CMLEs from protocatechuate-degrading organisms.
The sequence comparisons demonstrated that the two 3-sulfomuconate-converting CMLEs investigated in this study are more closely related to each other than to the corresponding enzymes from the protocatechuate pathways of various other bacteria. This was especially evident for ArCMLE2 from A. radiobacter S2 which is more closely related to the isofunctional HiCMLE2 from the taxonomically distantly related Hydrogenophaga strain (these organisms belong to the Alpha- or Betaproteobacteria) than to the previously described CMLEs from the protocatechuate pathways of other agrobacteria (Parke, 1996; Wood et al., 2001). This clearly suggested a horizontal transfer of the gene encoding ArCMLE2 from a different genetic background to strain S2.
Unfortunately, only very limited information is available about the enzymic characteristics of bacterial CMLEs. This is mainly due to the commercial unavailability and instability of the enzyme substrate 3-carboxy-cis,cis-muconate. Thus, since the pioneering work of Ornston (1966), who described the first purification and characterization of a CMLE (from P. putida), no other bacterial CMLEs have been studied in any detail with regard to their enzymic characteristics.
It was a surprising observation that PpCMLE1, which belongs to the protocatechuate pathway of P. putida, was also able to convert 3-sulfomuconate. This suggested that for CMLEs, no strict separation into type 1 enzymes (which only accept carboxylated substrates) and type 2 enzymes (which convert carboxylated plus sulfonated substrates) can be used as previously suggested for protocatechuate-cleaving activities (Feigel & Knackmuss, 1993). Nevertheless, it is evident that there are huge differences in the specific activities of the different types of CMLEs for the turnover of 3-carboxy-cis,cis-muconate. The comparison of the data given by Ornston (1966) for the CMLE from P. putida (and also our own results obtained with the His-tagged enzyme variant PpCMLE1) with HiCMLE2 and ArCMLE2, which participate in the degradation of 4-sulfocatechol, showed that the CMLE from P. putida converted 3-carboxy-cis,cis-muconate with a much higher vmax and a slightly lower Km than the type 2 enzymes from H. intermedia S1 and A. radiobacter S2. The CMLE from P. putida therefore seems to be much better adapted to the turnover of 3-carboxy-cis,cis-muconate than the type 2 enzymes studied here.
The analysis of the turnover of 3-sulfomuconate by PpCMLE did not allow a reliable calculation of the kinetic constants because of the observed substrate inhibition kinetics and the inherent difficulties of the HPLC test applied. Nevertheless, it became evident that the relative activities of this enzyme with 3-carboxy-cis,cis-muconate in comparison to the turnover of 3-sulfomuconate are much higher than the corresponding values for HiCMLE2 and ArCMLE2. Surprisingly, it appears that the specific activities of the purified PpCMLE with 3-sulfomuconate are higher than those determined for HiCMLE2 and ArCMLE2. This indicates that, for the turnover of 3-sulfomuconate from the point of enzyme specificity and activity, no specifically adapted CMLEs are necessary. To analyse this apparent contradiction more accurately, we are currently studying the turnover of 3-sulfomuconate by other CMLEs originating from the protocatechuate pathways of different bacteria.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 May 2006;
revised 20 July 2006;
accepted 20 July 2006.
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