Previous Article | Next Article 
Applied and Environmental Microbiology, December 1999, p. 5242-5246, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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 Dübendorf, Switzerland
Received 10 May 1999/Accepted 14 September 1999
 |
ABSTRACT |
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
(+)-cis-(1R,2S) enantiomer was
oxidized; the (
)-cis-(S,2R)
enantiomer remained unchanged. CDD oxidized both enantiomers of
cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but
oxidation of the (+)-cis-(1S,2R)
enantiomer was delayed until the
()-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 (+)-(1S) enantiomer to
1-indanone. This stereoselective transformation shows that CDD also
acted as an alcohol dehydrogenase. Additionally, CDD was able to
oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene,
(+)-cis-monochlorobiphenyl dihydrodiols, and
(+)-cis-toluene dihydrodiol to the corresponding catechols.
| |
INTRODUCTION |
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
cis-dihydrodiols 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 cis-benzene 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-3-phenylcyclohexa-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
cis-chlorobenzene 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.
| |
MATERIALS AND METHODS |
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 produced 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-µm-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
OD
578 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-(1
S,2
R)-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-1-methylcyclohexa-4,6-diene
(
cis-4-chlorotoluene
dihydrodiol),
cis-4-bromo-2,3-dihydroxy-1-methylcyclohexa-4,6-diene
(
cis-4-bromotoluene dihydrodiol),
cis-1,2-indandiol, 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-µm-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 (Macherey-Nagel, Düren, Germany). The system was operated
isocratically with a flow rate of 0.5 ml/min, and the injection volume
was 20 or 50 µl. The elution conditions were as follows:
cis-1,2-dihydroxy-1,2-dihydronaphthalene and
cis-1,2-dihydroxy-1,2,3,4-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-4-fluorotoluene 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 µl.
(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 µl of
N,N-dimethylformamide. One hundred microliters of
a solution of recrystallized n-butylboronic acid (500 µg
of n-butylboronic acid/ml 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 µl) 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 µl of ethyl acetate.
N,
O-bis(trimethylsilyl)trifluoroacetamide
(100 µl) 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 µl) 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 = (A
1 A
2)/(A
1 + A
2), where
A
1 and A
2 were the peak areas of the BB
derivatives of the
two
cis-dihydrodiol enantiomers,
respectively, and A
1 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 · 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).
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 1.
Enantioselective dehydrogenation of indandiol
enantiomers by whole cells of E. coli DH5(pTCB149)
expressing tcbB.
(+)-cis-(1R,2S)-Indandiol ( ) and
()-cis-(1S,2R)-indandiol ( ) were
produced by incubation of indan with E. coli DH5(pTCB144)
and were incubated together in a single reaction.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
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.
|
|
In contrast to
cis-1,2-indandiol, CDD dehydrogenated both
enantiomers of
cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (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.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 3.
Dehydrogenation of
(+)-cis(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene
(),
()-cis(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene
( ), and
(+)-cis(1R,2S)-dihydroxy-1,2-dihydronaphthalene
() 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.
|
|
(+)-
cis-(1
R,2
S)-Dihydroxy-1,2-dihydronaphthalene
(naphthalene dihydrodiol; ee > 98%) was transformed by CDD with
a rate of
16.3 nmol/min · 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.
Transformation of para-substituted toluene
dihydrodiols.
The absolute configuration of the enantiomer of
cis-4-chloro-2,3-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-dihydroxy-1-methylcyclohexa-4,6-diene (cis-4-bromotoluene
dihydrodiol), and
cis-4-iodo-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (cis-4-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-6-methylcatechol, 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-bromo-6-methylcatechol,
respectively. The isotope distribution was
also in accordance
with the proposed structures. No other products were
detected
by GC-MS and HPLC analyses.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 4.
Dehydrogenation of the major () and minor ()
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-4-fluorotoluene dihydrodiol; (B)
cis-4-chlorotoluene dihydrodiol; (C)
cis-4-iodotoluene dihydrodiol; (D)
cis-4-bromotoluene dihydrodiol.
|
|
Transformation of (+)-cis-toluene dihydrodiol and
cis-monochlorobiphenyl 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 · mg of
protein. Besides 3-methylcatechol, no other transformation products
were detected.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 5.
Transformation of (+)-cis-toluene dihydrodiol
() and concomitant formation of 3-methylcatechol () during
incubations with E. coli DH5(pTCB149).
|
|
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-2-chlorobiphenyl 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 (+)-(1S)-indanol.
Time course
studies with (±)-1-indanol as a substrate showed that CDD
enantioselectively and stoichiometrically dehydrogenated (+)-(1S)-indanol to 1-indanone (Fig.
6).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 6.
Enantioselective dehydrogenation of
(+)-(1S)-indanol () but not of
()-(1S)-indanol () to 1-indanone ( ) by whole cells
of E. coli DH5(pTCB149). Racemic indanol was incubated in
a single reaction.
|
|
| |
DISCUSSION |
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-1,2,3,4-tetrahydronaphthalene 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 five-membered, 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-4-chloro-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-2,3-dihydroxy-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-1,2-indandiol, 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 standards and motivating discussions, and we are
grateful to A. J. B. Zehnder for critical discussions and
comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, EAWAG, Überlandstrasse 133, CH-8600
Dübendorf, Switzerland. Phone: 41 1 823 5521. Fax: 41 1 823 5547. E-mail: kohler{at}eawag.ch.
Present address: Fichtner, 70191 Stuttgart, Germany.
| |
REFERENCES |
| 1.
|
Allen, C. C. R.,
D. R. Boyd,
H. Dalton,
N. D. Sharma,
I. Brannigan,
N. A. Kerley,
G. N. Sheldrake, and S. C. Taylor.
1995.
Enantioselective bacterial biotransformation routes to cis-diol metabolites of monosubstituted benzenes, naphthalene and benzocycloalkanes of either absolute configuration.
J. Chem. Soc. Chem. Commun.
1995:117-118.
|
| 2.
|
Boyd, D. R.,
N. D. Sharma,
M. V. Hand,
M. R. Groocock,
N. A. Kerley,
H. Dalton,
J. Chima, and G. N. Sheldrake.
1993.
Stereodirecting substituent effects during enzyme-catalysed synthesis of cis-dihydrodiol metabolites of 1,4-disubstituted benzene substrates.
J. Chem. Soc. Chem. Commun.
1993:974-976.
|
| 3.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 4.
|
Brand, J. M.,
D. L. Cruden,
G. J. Zylstra, and D. T. Gibson.
1992.
Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.
Appl. Environ. Microbiol.
58:3407-3410[Abstract/Free Full Text].
|
| 5.
|
Connors, N.,
R. Prevoznak,
M. Chartrain,
J. Reddy,
R. Singhvi,
Z. Patel,
R. Olewinski,
P. Salomon,
J. Wilson, and R. Greasham.
1997.
Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1.
J. Ind. Microbiol.
18:353-359.
|
| 6.
|
Eaton, S. L.,
S. M. Resnick, and D. T. Gibson.
1996.
Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains.
Appl. Environ. Microbiol.
62:4388-4394[Abstract].
|
| 7.
|
Gibson, D. T.,
D. L. Cruden,
J. D. Haddock,
G. J. Zylstra, and J. M. Brand.
1993.
Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707.
J. Bacteriol.
175:4561-4564[Abstract/Free Full Text].
|
| 8.
|
Gibson, D. T.,
M. Hensley,
H. Yoshioka, and T. J. Mabry.
1970.
Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida.
Biochemistry.
9:1626-1630[Medline].
|
| 9.
|
Gibson, D. T.,
J. R. Koch,
C. L. Shuld, and R. E. Kallio.
1968.
Oxidative degradation of aromatic hydrocarbons by microorganisms. II. Metabolism of halogenated aromatic hydrocarbons.
Biochemistry
7:3795-3802[Medline].
|
| 10.
|
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[Abstract/Free Full Text].
|
| 11.
|
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
|
| 12.
|
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[Medline].
|
| 13.
|
Patel, T. R., and D. T. Gibson.
1974.
Purification and properties of (+)-cis-naphthalene dihydrodiol dehydrogenase of Pseudomonas putida.
J. Bacteriol.
119:879-888[Abstract/Free Full Text].
|
| 14.
|
Raschke, H.
1998.
Ph.D. thesis.
Swiss Federal Institute of Technology, Zürich, Switzerland
|
| 15.
|
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.
|
| 16.
|
Rogers, J. E., and D. T. Gibson.
1977.
Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida.
J. Bacteriol.
130:1117-1124[Abstract/Free Full Text].
|
| 17.
|
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
|
| 18.
|
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[Abstract/Free Full Text].
|
| 19.
|
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[Medline].
|
Applied and Environmental Microbiology, December 1999, p. 5242-5246, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Jouanneau, Y., Meyer, C.
(2006). Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols.. Appl. Environ. Microbiol.
72: 4726-4734
[Abstract]
[Full Text]
-
Pollmann, K., Beil, S., Pieper, D. H.
(2001). Transformation of Chlorinated Benzenes and Toluenes by Ralstonia sp. Strain PS12 tecA (Tetrachlorobenzene Dioxygenase) and tecB (Chlorobenzene Dihydrodiol Dehydrogenase) Gene Products. Appl. Environ. Microbiol.
67: 4057-4063
[Abstract]
[Full Text]
-
Parales, R. E., Resnick, S. M., Yu, C.-L., Boyd, D. R., Sharma, N. D., Gibson, D. T.
(2000). Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene cis-Dihydroxylation: Control by Phenylalanine 352 in the alpha Subunit. J. Bacteriol.
182: 5495-5504
[Abstract]
[Full Text]
Top
Abstract
Introduction
