May 10, 1999 - cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from. Pseudomonas sp. ... CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1999, p. 5242–5246 0099-2240/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 65, No. 12
cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5␣(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions HENNING RASCHKE,† THOMAS FLEISCHMANN, JAN ROELOF VAN DER MEER, AND HANS-PETER E. KOHLER* Swiss Federal Institute for Environmental Sciences and Technology (EAWAG), CH-8600 Du ¨bendorf, Switzerland Received 10 May 1999/Accepted 14 September 1999
cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5␣(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (ⴙ)-cis-(1R,2S) enantiomer was oxidized; the (ⴚ)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4tetrahydronaphthalene, but oxidation of the (ⴙ)-cis-(1S,2R) enantiomer was delayed until the (ⴚ)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (ⴙ)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (ⴙ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (ⴙ)-cis-monochlorobiphenyl dihydrodiols, and (ⴙ)-cis-toluene dihydrodiol to the corresponding catechols. The aerobic bacterial degradation of nonactivated aromatic compounds is usually initiated by dioxygenases that incorporate two hydroxyl groups into the aromatic substrate. The products of such reactions are chiral cis-dihydrodiols with two adjacent stereogenic centers. Dioxygenases that act on aromatic compounds generally are broad-spectrum enzymes (11). They stereoselectively oxidize some of their substrates to enantiomerically pure dihydrodiols or alcohols (7, 14, 15); this stereoselectivity led to an interest in the use of such enzymes for the production of chiral synthons. With other substrates, the stereospecificity of the reaction is relaxed and the products are nonracemic mixtures of enantiomers (14, 15). During the next step in the degradation of aromatic compounds, the cisdihydrodiols are dehydrogenated to catechols by cis-dihydrodiol dehydrogenases. Generally, cis-dihydrodiol dehydrogenases are also broad-substrate enzymes, and most dehydrogenases are able to transform several cis-dihydrodiol isomers (1, 6, 12, 13, 18). However, all studies that investigated the stereochemistry of cis-dihydrodiol oxidation, with the exception of one study (16), report that cis-dihydrodiol dehydrogenases catalyze stereoselective transformations. For example, purified naphthalene dihydrodiol dehydrogenase from Pseudomonas sp. strain 119 enantioselectively oxidizes polycyclic aromatic compounds with a cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene residue and monosubstituted cis-benzene dihydrodiols with an S configuration at the C atom in meta position with respect to the substituent (12, 13). Whole cells of Pseudomonas putida NCIMB 8859, which contain a cis-dihydrodiol dehydrogenase, exclusively transform the cis-(1R,2S) enantiomers of cis-1,2-
dihydroxy-1,2-dihydronaphthalene, cis-1,2-indandiol, cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, and cis-1,2-dihydroxybenzocycloheptane but not the cis-(1S,2R) enantiomers (1). The same strain preferentially transforms monosubstituted cisbenzene dihydrodiols with an S configuration at the C atom in meta position with respect to the substituent. The cis-biphenyl dihydrodiol dehydrogenase from Sphingomonas yanoikuyae B1 oxidizes both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene but only the (1S,2R) enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3phenylcyclohexa-3,5-diene (6). The cis-glycol dehydrogenase of P. putida 421-5 (ATCC 55687) enantioselectively transforms cis-(1R,2S)-indandiol (5). This project was started to investigate the potential of cischlorobenzene dihydrodiol dehydrogenase (CDD) for enantiomeric resolution of chiral dihydrodiols. Previously, we have shown that the cis-dihydrodiols formed by chlorobenzene dioxygenase (CDO) of Pseudomonas sp. strain P51 from biphenyl, naphthalene, toluene, and 1,2-dichlorobenzene are oxidized to the corresponding catechols when incubated with the recombinant Escherichia coli DH5␣(pTCB149) (18). The strain DH5␣(pTCB149) expresses the CDD gene (tcbB) of Pseudomonas sp. strain P51. Here, we present data on stereoselective transformations of nonracemic mixtures of various dihydrodiols upon incubation with E. coli DH5␣(pTCB149) and show that CDD preferentially oxidized those para-halogenated cis-toluene dihydrodiol enantiomers that were also preferentially formed by CDO.
* Corresponding author. Mailing address: Department of Microbi¨ berlandstrasse 133, CH-8600 Du ology, EAWAG, U ¨bendorf, Switzerland. Phone: 41 1 823 5521. Fax: 41 1 823 5547. E-mail: kohler@eawag .ch. † Present address: Fichtner, 70191 Stuttgart, Germany.
Chemicals. Standards of (⫾)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (⫹)-cis-(1R,2S)-indandiol (enantiomeric excess [ee] of about 70%) (10) were gifts from S. M. Resnick and D. T. Gibson (University of Iowa). (⫹)-cis-(1R,2S)Indandiol (ee ⫽ 30%), (⫹)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (ee ⬎ 98%), (⫹)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene (ee ⫽ 82%), (⫹)-cis-toluene dihydrodiol, and cis-monochlorobiphenyl dihydrodiols were pro-
MATERIALS AND METHODS
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duced with E. coli DH5␣(pTCB144). For the production of para-substituted nonracemic cis-toluene dihydrodiols, we used P. putida F39/D or E. coli DH5␣(pTCB144). cis-4-Bromotoluene dihydrodiol, cis-4-chlorotoluene dihydrodiol, and cis-4-iodotoluene dihydrodiol were produced with P. putida F39/D; cis-4-fluorotoluene dihydrodiol was produced with E. coli DH5␣(pTCB144). All the other chemicals were obtained from Fluka (Buchs, Switzerland). Microorganisms, growth conditions, and biotransformation procedure. E. coli DH5␣(pTCB144) containing the CDO genes, E. coli DH5␣(pTCB149) containing the CDD gene, and E. coli DH5␣(pUC18) containing the cloning vector were grown at 25°C on Luria-Bertani medium in the presence of 100 mg of ampicillin/ liter (18). After approximately 36 h, the cells had reached an optical density at 578 nm (OD578) of around 3.5. They were then harvested, washed, and resuspended in M9 mineral medium (17) with 1 mM glucose. The OD578 was adjusted to approximately 1.0. P. putida F39/D was kindly provided by D. T. Gibson and S. M. Resnick. F39/D is a mutant of P. putida F1 that lacks cis-dihydrodiol dehydrogenase activity (8, 19). It was grown at 30°C in M9 medium (17) with 5 mM pyruvate in the presence of toluene in the headspace. After 30 h, the cells had reached an OD578 of about 3.0. The cells were then harvested, washed, and resuspended in M9 mineral medium with 5 mM pyruvate. The OD578 was adjusted to 1.0. For the production of cis-dihydrodiols, the biotransformation substrates were added at 1% (vol/vol) from stock solutions in methanol (100 mM) to give final concentrations of 1.0 mM. Biotransformations with E. coli DH5␣(pTCB144) were performed at 25°C, and those with P. putida F39/D were performed at 30°C. After the formation of the cis-dihydrodiols, the media were centrifuged at 8,000 rpm for 8 min and the supernatants were filtered through 0.20-m-pore-size Schleicher & Schuell (Dassel, Germany) FP 030/3 filters in order to remove the production strains. The filtered supernatants were frozen and stored at ⫺18°C until they were used for incubations with E. coli DH5␣(pTCB149). Filtered biotransformation media from incubations with P. putida F39/D and E. coli DH5␣(pTCB144) were free of toluene dioxygenase and CDO activity, since incubations of the filtered medium with 4-bromotoluene (1% [vol/vol] dissolved in methanol; concentration of stock solution, 100 mM) did not lead to any detectable cis-dihydrodiol products by gas chromatography-mass spectrometry (GC-MS) or high-performance liquid chromatography (HPLC) analysis. For biotransformation experiments with E. coli DH5␣(pTCB149), 25 ml of the cell suspension (adjusted to an OD578 of 3.0 after washing) was supplemented with 50 ml of the filtered supernatants from the biotransformation with E. coli DH5␣(pTCB144) or P. putida F39/D. For the experiment with 1-indanol, 25 ml of E. coli DH5␣(pTCB149) cell suspension was supplemented with 50 ml of M9 mineral medium, which was previously spiked with 0.75 ml of (⫾)-1-indanol (100 mM in methanol). The final concentration of (⫾)-1-indanol in the biotransformation experiment was 1.0 mM. To confirm CDD activity (positive control), E. coli DH5␣(pTCB149) cultures were incubated with (⫹)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene [(⫹)-cis-toluene dihydrodiol]. In such experiments, the formation of 3-methylcatechol was followed by HPLC and GC-MS analysis. Negative-control experiments were done with E. coli DH5␣(pUC18), which only contained the cloning vector and thus did not express CDO or CDD activity (18). No transformation products and no decrease in the concentrations of cis-4-chloro-2,3-dihydroxy-1methylcyclohexa-4,6-diene (cis-4-chlorotoluene dihydrodiol), cis-4-bromo-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (cis-4-bromotoluene dihydrodiol), cis-1,2indandiol, and 1-indanol were detected by GC-MS and HPLC analysis. Analytical procedures. (i) HPLC. Samples (2 ml) from the biotransformation incubations were filtered through 0.20-m-pore-size Schleicher & Schuell FP 030/3 filters or centrifuged at 16,000 ⫻ g for 2 min. They were then analyzed on a 625 LC HPLC system equipped with a WISP 700 autosampler and a 901 photodiode array detector (Waters Millipore Corp., Milford, Mass.). Samples of the incubations with cis-toluene dihydrodiol were acidifed to pH 1 in order to dehydrate cis-toluene dihydrodiol to ortho-cresol, which was then quantified by HPLC analysis. Separation was done on a C18 reverse-phase column (MachereyNagel, Du ¨ren, Germany). The system was operated isocratically with a flow rate of 0.5 ml/min, and the injection volume was 20 or 50 l. The elution conditions were as follows: cis-1,2-dihydroxy-1,2-dihydronaphthalene and cis-1,2-dihydroxy1,2,3,4-tetrahydronaphthalene were separated with an eluent consisting of 20% (vol/vol) eluent A (10 mM H3PO4, pH 3.0) and 80% eluent B (90% methanol and 10% eluent A). cis-1,2-Indandiol, cis-4-chlorotoluene dihydrodiol, cis-4fluorotoluene dihydrodiol, cis-4-bromotoluene dihydrodiol, cis-4-iodotoluene dihydrodiol, ortho-cresol, and 3-methylcatechol were analyzed with an eluent consisting of 50% (vol/vol) eluent B and 50% eluent C (90% methanol and 10% distilled water). (⫾)-1-Indanol was analyzed on a Chiralcel OD-R column (Daicel Chemical Industries, Tokyo, Japan) on an HPLC system with a photodiode array detector (Gynkotek GmbH, Germering, Germany). The eluent consisted of 90% (vol/vol) 0.5 M HClO4, pH 2.0, and 10% acetonitrile. The Gynkotek system was operated isocratically with a flow rate of 0.7 ml/min, and the injection volume was 150 l. (ii) GC-MS analysis. For GC-MS analysis, dihydrodiols were extracted from 5 to 10 ml of the supernatants of the incubation mixtures with equal volumes of ethyl acetate. The ethyl acetate extract was dried with sodium sulfate and evaporated to dryness under a gentle stream of nitrogen at 40°C. The residue was dissolved in 100 l of N,N-dimethylformamide. One hundred microliters of a solution of recrystallized n-butylboronic acid (500 g of n-butylboronic acid/ml
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FIG. 1. Enantioselective dehydrogenation of indandiol enantiomers by whole cells of E. coli DH5␣(pTCB149) expressing tcbB. (⫹)-cis-(1R,2S)-Indandiol (E) and (⫺)-cis-(1S,2R)-indandiol (䊐) were produced by incubation of indan with E. coli DH5␣(pTCB144) and were incubated together in a single reaction.
dissolved in N,N-dimethylformamide) was added, and the mixture was heated to 70°C for 15 min to form the n-butylboronate (BB) derivatives. Samples of the BB derivatives were diluted with cyclohexane (at least 15-fold). Separation and quantification of the enantiomers was achieved by GC-MS analysis. A Tribrid double-focusing magnetic-sector hybrid mass spectrometer (VG Analytical, Manchester, England) was used. The enantiomers were separated as their BB derivatives on a 25% t-butyldimethylsilylated--cyclodextrin column (30 by 0.25 by 0.25 m), obtained from BGB Analytik AG, Rothenfluh, Switzerland. Diluted samples (0.5 l) were injected on the column at 60°C. The column temperature was programmed as follows: 15°C/min to 160°C, 3°C/min to 230°C, and 20°C/min to 250°C. All samples were analyzed by electron ionization (70 eV) with full-scan monitoring (m/z ⫽ 50 to 250 or m/z ⫽ 50 to 400). In order to generate trimethylsilyl (TMS) derivatives of catechols, metabolites were extracted as described above. The residue was dissolved in 100 l of ethyl acetate. N,O-bis(trimethylsilyl)trifluoroacetamide (100 l) was added, and the mixture was heated to 70°C for 15 min to generate TMS derivatives. Mass spectra were obtained with an ITD 800 (ion trap detection) mass spectrometer (Finnigan MAT, San Jose, Calif.) coupled to an HRGC 5160 Mega Series gas chromatograph (Carlo Erba Instruments, Milan, Italy) equipped with a 10-m PS090 (80% dimethyl, 20% diphenyl) glass capillary column. Electron ionization (70 eV) was used. The injection (0.5 l) occurred on column at 50°C. The temperature was programmed from 50 to 250°C at 10°C/min. The ee was defined as follows: ee ⫽ (A1 ⫺ A2)/(A1 ⫹ A2), where A1 and A2 were the peak areas of the BB derivatives of the two cis-dihydrodiol enantiomers, respectively, and A1 was the peak with the larger area. (iii) Protein determination. Protein contents were determined by the method of Bradford (3) with bovine serum albumin as the standard.
RESULTS Transformation of benzocyclic dihydrodiol substrates. Incubation of a nonracemic mixture of cis-1,2-indandiol with E. coli DH5␣(pTCB149) led to the transformation of the (⫹)-cis(1R,2S)-enantiomer (Fig. 1) at a rate of 5.3 nmol/min 䡠 mg of protein. The (⫺)-cis-(1S,2R)-enantiomer was not converted (Fig. 1). The products of the dehydrogenation were 1,2-indenediol and a monohydroxylated compound. The identity of 1,2-indenediol was confirmed by GC-MS analysis of the TMS derivative (molecular ion m/z, 292). Ions generated by the loss of one of the methyl groups of the TMS moiety (m/z, 277; M-15) and by the loss of a TMS group (m/z, 219; M-73) were in agreement with the proposed structure of the dehydrogenation product. The monohydroxylated product was also characterized by GC-MS analysis of its TMS derivative (molecular ion m/z, 220). The spectrum was dominated by ions at an m/z of 205 (loss of one of the methyl groups of the TMS moiety) and at an m/z of 147 (loss of TMS). This product most likely is ketohydroxy indan, which is easily formed by tautomerization of 1,2-indenediol (5) (Fig. 2). In contrast to cis-1,2-indandiol, CDD dehydrogenated both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphtha-
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FIG. 2. Proposed oxidation of (⫹)-cis)-(1R,2S)-indandiol by E. coli DH5␣(pTCB149) to 1,2-indenediol and subsequent chemical tautomerization of 1,2-indenediol to ketohydroxy indan.
lene (Fig. 3). However, the (⫹) enantiomer was turned over faster than the (⫺) enantiomer. The formation of 1,2-dihydroxy-3,4-dihydronaphthalene as the product of this reaction was confirmed by GC-MS analysis of its TMS derivative (molecular ion m/z, 306). Ions generated by the loss of one of the methyl groups of the TMS moiety (m/z, 291; M-15), by the loss of one of the TMS groups (m/z, 233; M-73), by the loss of both TMS groups (m/z, 162; M-146), and by the loss of one of the TMS groups and an OTMS group (m/z, 144; M-162) are in accordance with the proposed structure of the dehydrogenation product. (⫹)-cis-(1R,2S)-Dihydroxy-1,2-dihydronaphthalene (naphthalene dihydrodiol; ee ⬎ 98%) was transformed by CDD with a rate of 16.3 nmol/min 䡠 mg of protein (Fig. 3). The incubation medium became yellow after an incubation period of 30 min, which was most likely due to the auto-oxidation of 1,2-dihydroxynaphthalene to 1,2-naphthoquinone (13). This also explains why the proposed dehydrogenation product, 1,2-dihydroxynaphthalene, could not be detected by GC-MS analysis.
FIG. 3. Dehydrogenation of (⫹)-cis(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene (䊐), (⫺)-cis(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene (‚), and (⫹)-cis(1R,2S)-dihydroxy-1,2-dihydronaphthalene (E) by whole cells of E. coli DH5␣(pTCB149). The substrates were produced by incubation of 1,2-dihydronaphthalene with E. coli DH5␣(pTCB144) and were incubated together in a single reaction.
FIG. 4. Dehydrogenation of the major (E) and minor (䊐) enantiomers of nonracemic mixtures of para-substituted cis-toluene dihydrodiols by E. coli DH5␣(pTCB149). Dihydrodiols were produced by P. putida F39/D or E. coli DH5␣(pTCB144) from the corresponding para-substituted toluenes. (A) cis-4fluorotoluene dihydrodiol; (B) cis-4-chlorotoluene dihydrodiol; (C) cis-4-iodotoluene dihydrodiol; (D) cis-4-bromotoluene dihydrodiol.
Transformation of para-substituted toluene dihydrodiols. The absolute configuration of the enantiomer of cis-4-chloro2,3-dihydroxy-1-methylcyclohexa-4,6-diene (cis-4-chlorotoluene dihydrodiol) that is preferentially produced by the TDO of P. putida F39/D is unknown but identical with the absolute configuration of the enantiomer that is preferentially produced by CDO (14). This is also true for cis-4-bromo-2,3-dihydroxy1-methylcyclohexa-4,6-diene (cis-4-bromotoluene dihydrodiol), and cis-4-iodo-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (cis4-iodotoluene dihydrodiol) (14). To distinguish between the two enantiomers, they are called the major and minor enantiomer in the following discussion. The major enantiomer is the enantiomer which is preferentially formed by TDO and CDO. CDD oxidized both enantiomers of para-substituted cis-toluene dihydrodiols (Fig. 4). It can be seen that the major enantiomers were oxidized by CDD at higher rates than the minor enantiomers. The transformation of the two enantiomers of cis-4-fluorotoluene dihydrodiol, cis-4-chlorotoluene dihydrodiol, and cis-4-iodotoluene dihydrodiol started at the same time (Fig. 4A to C). Transformation of the minor enantiomer of cis-4-bromotoluene dihydrodiol started after the major enantiomer was completely depleted (Fig. 4D). GC-MS analysis of the TMS derivatives of the transformation products showed the formation of the respective catechols in each experiment (3-fluoro-6-methylcatechol, molecular ion m/z, 286; 3-chloro-6-methylcatechol, molecular ion m/z, 302; 3-bromo-6methylcatechol, molecular ion m/z, 346; and 3-iodo-6-methylcatechol, molecular ion m/z, 394). The identities of the catechol products were also confirmed by their fragmentation patterns. Ions generated by the loss of a methyl group (M-15), a TMS group (M-73), and an OTMS group (M-89) and ions at an m/z of 73 and 89 (TMS and OTMS) were observed in all spectra. The loss of chlorine (M-35) or bromine (M-79) was seen in the spectra of 3-chloro-6-methylcatechol and 3-bromo6-methylcatechol, respectively. The isotope distribution was
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electively and stoichiometrically dehydrogenated (⫹)-(1S)-indanol to 1-indanone (Fig. 6). DISCUSSION
FIG. 5. Transformation of (⫹)-cis-toluene dihydrodiol (E) and concomitant formation of 3-methylcatechol (䊐) during incubations with E. coli DH5␣ (pTCB149).
also in accordance with the proposed structures. No other products were detected by GC-MS and HPLC analyses. Transformation of (ⴙ)-cis-toluene dihydrodiol and cismonochlorobiphenyl dihydrodiols. CDD oxidized (⫹)-cis-toluene dihydrodiol stoichiometrically to 3-methylcatechol (Fig. 5). The consumption rate of (⫹)-cis-toluene dihydrodiol and the building rate of 3-methylcatechol were 34.1 nmol/min 䡠 mg of protein. Besides 3-methylcatechol, no other transformation products were detected. As shown in another study (14), CDO oxidizes monochlorobiphenyls to pure cis-dihydrodiol enantiomers. These dihydrodiols were used as biotransformation substrates for CDD. CDD oxidized (⫹)-cis-2⬘,3⬘-dihydroxy-2⬘,3⬘-dihydro-2chlorobiphenyl to 2,3-dihydroxy-2⬘-chlorobiphenyl, oxidized (⫹)-cis-2⬘,3⬘-dihydroxy-2⬘,3⬘-dihydro-3-chlorobiphenyl to 2,3-dihydroxy-3⬘-chlorobiphenyl, and oxidized (⫹)-cis-2⬘,3⬘-dihydroxy-2⬘,3⬘-dihydro-4-chlorobiphenyl to 2,3-dihydroxy-4⬘-chlorobiphenyl. The formation of the catechol products was confirmed by the GC-MS analysis of their TMS derivatives (molecular ion m/z, 364). The ions at an m/z of 329 (loss of chlorine), an m/z of 291 (loss of TMS), an m/z of 276 (loss of TMS and of one methyl group of the other TMS group), an m/z of 180 (loss of TMS and of the chlorinated phenyl ring), and an m/z of 73 (TMS) dominated the three spectra and were in agreement with the proposed structures. Besides catechols, no other transformation products were detected by GC-MS analysis. Transformation of (ⴙ)-(1S)-indanol. Time course studies with (⫾)-1-indanol as a substrate showed that CDD enantios-
FIG. 6. Enantioselective dehydrogenation of (⫹)-(1S)-indanol (E) but not of (⫺)-(1S)-indanol (䊐) to 1-indanone ({) by whole cells of E. coli DH5␣(pTCB149). Racemic indanol was incubated in a single reaction.
In this study we showed that CDD was able to transform several cis-dihydrodiols to their respective catechols. The enantioselectivity of CDD was examined with benzocyclic and para-substituted cis-toluene dihydrodiols. The results of the experiments with benzocyclic cis-dihydrodiols showed that CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4tetrahydronaphthalene but only the (⫹)-cis-(1R,2S)-enantiomer of cis-1,2-indandiol (Fig. 1 to 3). We assume that CDD preferentially oxidized benzocyclic cis-dihydrodiols with cis(1S,2R) configurations. It tolerated six-membered, but not fivemembered, rings of the opposite absolute configuration. This assumption was supported by the observation that CDD enantioselectively oxidized (⫹)-(1S)-indanol (Fig. 6), which has the same spatial configuration at the C-1 position as (⫹)-cis(1R,2S)-indandiol. CDD dehydrogenated both enantiomers of para-substituted cis-toluene dihydrodiols. The major enantiomer formed by CDO or TDO was always transformed faster by CDD than the corresponding minor enantiomer (Fig. 4). CDD seems to be best adapted to those enantiomers that are preferentially formed by CDO. The absolute configuration of the cis-4chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene that is preferentially formed by dioxygenases in incubations with Pseudomonas strains is somewhat controversial. Gibson et al. (9) reported preferential formation of the (⫹)-cis-4-chloro-2,3dihydroxy-1-methylcyclohexa-4,6-diene in incubations with P. putida grown on toluene, whereas Boyd et al. (2) reported the preferential formation of the (⫺)-(2S,3S) enantiomer in incubations with P. putida UV4. As we did not have enough material available for measuring the optical rotation, we cannot infer the absolute configuration of the enantiomers that are preferentially formed and transformed by CDO and CDD, respectively. We cannot exclude the possibility that other products than catechols were formed in some of our incubations, since we were not able to establish exact mass balances due to the lack of authentic standards. When authentic standards of the products were available (1-indanone and 3-methylcatechol), an exact mass balance was obtained; we found no indication of additional products. With respect to enantioselective transformation of cis-1,2indandiol, CDD closely resembles the cis-dihydrodiol dehydrogenase of P. putida NCIMB 8859 (1) but differs from the cis-glycol dehydrogenase of P. putida 421-5. P. putida NCIMB 8859 enantioselectively oxidizes the (⫺)-cis-(1R,2S)-enantiomer (1), whereas the cis-biphenyl dihydrodiol dehydrogenase of S. yanoikuyae B1 only oxidizes the (⫹)-cis-(1S,2R) enantiomer (6). CDD converted (⫹)-(1S)-indanol stoichiometrically to 1-indanone (Fig. 6). This finding shows for the first time that a cis-dihydrodiol dehydrogenase can also act as an alcohol dehydrogenase. In P. putida F39/D (4), which lacks the dihydrodiol dehydrogenase, a similar reaction has been found and was ascribed to the presence of a 1-indanol dehydrogenase that preferentially oxidizes (⫹)-(1S)-indanol. Our results show that CDD can be used successfully for the resolution of chiral indanol. ACKNOWLEDGMENTS We thank M. Suter for help with GC-MS analysis, D. T. Gibson for providing P. putida F39/D, and S. M. Resnick for providing dihydrodiol
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standards and motivating discussions, and we are grateful to A. J. B. Zehnder for critical discussions and comments on the manuscript.
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10.
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degradation of aromatic hydrocarbons by microorganisms. II. Metabolism of halogenated aromatic hydrocarbons. Biochemistry 7:3795–3802. Gibson, D. T., S. M. Resnick, K. Lee, J. M. Brand, D. S. Torok, L. P. Wackett, M. J. Schocken, and B. E. Haigler. 1995. Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4. J. Bacteriol. 177:2615–2621. Gibson, D. T., and V. Subramanian. 1984. Microbial degradation of aromatic hydrocarbons, p. 181–252. In D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker, Inc., New York, N.Y. Jeffrey, A. M., H. J. C. Yeh, D. M. Jerina, T. R. Patel, J. F. Davey, and D. T. Gibson. 1975. Initial reactions in the oxidation of naphthalene by Pseudomonas putida. Biochemistry 14:575–584. Patel, T. R., and D. T. Gibson. 1974. Purification and properties of (⫹)-cisnaphthalene dihydrodiol dehydrogenase of Pseudomonas putida. J. Bacteriol. 119:879–888. Raschke, H. 1998. Ph.D. thesis. Swiss Federal Institute of Technology, Zu ¨rich, Switzerland. Resnick, S. M., K. Lee, and D. T. Gibson. 1996. Diverse reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Ind. Microbiol. 17:438–457. Rogers, J. E., and D. T. Gibson. 1977. Purification and properties of cistoluene dihydrodiol dehydrogenase from Pseudomonas putida. J. Bacteriol. 130:1117–1124. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Werlen, C., H. P. E. Kohler, and J. R. van der Meer. 1996. The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J. Biol. Chem. 271:4009– 4016. Ziffer, H., D. M. Jerina, D. T. Gibson, and V. M. Kobal. 1973. Absolute stereochemistry of the (⫹)-cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene produced from toluene by Pseudomonas putida. J. Am. Chem. Soc. 95:4048– 4049.
