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Applied and Environmental Microbiology, May 2000, p. 2148-2153, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Fluoropyrogallols as New
Intermediates in Biotransformation of Monofluorophenols in
Rhodococcus opacus 1cp
Zoya I.
Finkelstein,1
Boris P.
Baskunov,1
Marelle G.
Boersma,2
Jacques
Vervoort,2
Eugene L.
Golovlev,1
Willem J. H.
van Berkel,2
Ludmila A.
Golovleva,1 and
Ivonne
M. C. M.
Rietjens2,*
G. K. Skrybin Institute of Biochemistry
and Physiology of Microorganisms, Russian Academy of Sciences,
Pushchino, Russia,1 and Department of
Biomolecular Sciences, Laboratory of Biochemistry, Wageningen
University, NL-6703 HA Wageningen, The
Netherlands2
Received 19 November 1999/Accepted 28 February 2000
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ABSTRACT |
The transformation of monofluorophenols by whole cells of
Rhodococcus opacus 1cp was investigated, with special
emphasis on the nature of hydroxylated intermediates formed. Thin-layer
chromatography, mass spectrum analysis, and 19F
nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three
monofluorophenols. The 19F chemical shifts and
proton-coupled splitting patterns of the fluorine resonances of the
trihydroxyfluorobenzene products established that the
trihydroxylated aromatic metabolites contained hydroxyl substituents on
three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from
2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene
(5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were
observed. These results indicate the involvement of fluoropyrogallols
as previously unidentified metabolites in the
biotransformation of monofluorophenols in R. opacus 1cp.
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INTRODUCTION |
Halophenols and their derivatives
are priority pollutants of mainly anthropogenic origin. Over several
decades, these compounds have been widely used as building blocks in
chemical and pharmaceutical syntheses and as herbicides and pesticides,
and they have caused serious local contamination of the environment.
Soil microorganisms have developed the capacity of utilizing
halophenols for their growth by a diverse set of biodegradation
pathways (8). Aerobic soil microorganisms generally
degrade mono- and dihalophenols through the initial action of
(chloro)phenol ortho-hydroxylases, leading to the formation
of halocatechols (1, 7, 9, 10, 12). In the framework of
a project devoted to the biodegradation of halophenols by
gram-positive bacteria, we investigated the formation of
hydroxylated intermediates formed upon the conversion of
halophenols by various Rhodococcus species and
previously demonstrated the formation of (halo)catechols as
initial intermediates in the biodegradation pathways (3).
However, identification of the subsequent biodegradation pathways of
the chlorocatechols appeared hampered by the fact that
unequivocal identification of the site of introduction of a
third hydroxyl group is difficult because 1H
nuclear magnetic resonance (NMR) splitting patterns combined with
1H chemical shift data of the protons present in
these metabolites can be compatible with more than one
substitution pattern (13). Therefore, in this paper, we have
studied the possible formation of trihydroxyfluorobenzene metabolites
from fluorophenols by whole cells of Rhodococcus opacus 1cp
in detail. The fluorine substituent provides the possibility to detect
and quantify the possible hydroxyfluorobenzene intermediates by
19F NMR, allowing the identification of the exact
substitution pattern. Using this technique we unambiguously demonstrate
the formation of fluoropyrogallols
(1,2,3-trihydroxyfluorobenzenes) as new intermediates in the
biotransformation of monofluorophenols by R. opacus 1cp.
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MATERIALS AND METHODS |
Chemicals.
Phenol was purchased from Merck (Darmstadt,
Germany). 2-Fluorophenol, 3-fluorophenol, and 4-fluorophenol were
purchased from Janssen Chimica (Beerse, Belgium). Fluorocatechols were
prepared from the corresponding fluorophenols using purified phenol
hydroxylase from Trichosporon cutaneum (14).
Fluoromuconates were prepared and identified as described previously
(2) by incubating the fluorocatechols with catechol
1,2-dioxygenase from Pseudomonas arvilla C-1.
Growth of R. opacus 1cp.
The strain R. opacus 1cp was isolated and maintained as described previously
(6). The strain can grow on phenol as the sole source of
carbon. For cultivation, a mineral synthetic medium containing, per
liter, 1 g of NH4NO3, 1 g of
K2HPO4, 1 g of
KH2PO4, 0.2 g of MgSO4
· 7H2O, 0.02 g of CaCl2, and 2 drops of
a saturated solution of FeCl3 (pH 7.2) was used. Phenol was
used as the source of carbon and was initially added at a 200-mg/liter
final concentration. R. opacus 1cp did not grow on either of
the three monofluorophenols as the sole source of carbon, and,
therefore, fluorophenols were added as inducers in addition to phenol.
After 6 h an additional portion of phenol (200 mg/liter) was added
together with 2 mg of the fluorophenol to be tested/liter. Cultures
were incubated at 28°C on an orbital shaker (2,000 rpm). After 20 to
22 h cell density was 0.24 to 0.36 at 540 nm. After 20 to 22 h of cultivation the cells were harvested by centrifugation (20 min,
5,000 × g) and washed twice in potassium phosphate, pH
7.0.
Incubation conditions.
Measurements using cell extracts of
R. opacus 1cp appeared to be hampered by the fact that the
phenol hydroxylases from the Rhodococcus species appear to
be highly labile (4, 11, 15, 18, 23). Thus,
biotransformation of the fluorophenols was investigated in incubations
with whole cells. To test the biotransformation of fluorophenols,
fresh, washed R. opacus 1cp cells, harvested from five to
seven flasks with 200 ml of culture liquids, were resuspended in
mineral medium without phenol (optical density, 2 to 3 at 540 nm). To
20 ml of this cell suspension the fluorophenol under investigation was
added to a final concentration of 1 mM, and the cultures were incubated
at 28°C on an orbital shaker. Each experiment was conducted in
triplicate, and control experiments were performed with medium without
cells or with medium without the corresponding phenol. To monitor the
conversion of substrates, every 0.5 h the metabolite profile in
one of the flasks was analyzed. To this end, the incubation mixture was
acidified with 1 N HCl to pH 2.0, followed by extraction with ethyl
acetate three times. These ethyl acetate fractions were concentrated by
evaporation under vacuum, and the samples thus obtained were analyzed
by thin-layer chromatography (TLC), mass spectrometry (MS), and/or
19F NMR.
TLC, HPLC, and MS analysis.
Qualitative analysis of
intermediates was performed by TLC on DC-AlufolienKieselgel 60 F254
plates (Merck) developed with benzene-dioxane-acetic acid (90:10:2).
After the processing of the plates, metabolites were detected under UV
light, with diazotized benzidine and AgNO3 in acetone
solution. After visualization, compounds with corresponding
Rf values were eluted from the plates with
methanol. Methanol extracts were evaporated, and the purity of the
compounds was checked by TLC. Pure compounds were analyzed on a
Finnigan MAT 8430 mass spectrometer operated at an ionization energy of
70 eV with direct evaporation of samples. For high-pressure liquid
chromatography (HPLC) analysis the culture liquid was acidified to pH
2.0 and extracted three times with ethyl acetate. HPLC analysis was
conducted using a reversed-phase column (4.0 by 250 mm; Spherisorb ODS-2,2134 LKB). Elution was carried out isocratically at a flow rate
of 0.8 ml min
1 with 1.5 mM KH2PO4
containing 30% methanol. Detection was at 280 nm. Metabolite peaks
were identified and quantified in comparison with standard compounds.
19F NMR measurements.
19F NMR
measurements were performed on Bruker AMX 300 and Bruker DPX 400 NMR
spectrometers as described previously (2, 21). The
temperature was 7°C unless indicated otherwise. A dedicated 10-mm
19F NMR probe head was used for both instruments. The
spectral width for the 19F NMR measurements was between
20,000 and 50,000 Hz depending on the sample of interest. The number of
data points used for data acquisition was 65,536. Pulse angles of 30°
were used. Between 2,000 and 60,000 scans were recorded, depending on
the concentrations of the fluorine-containing compounds and the
signal-to-noise ratio required. The detection limit of an overnight run
(60,000 scans) was 1 µM. The sample volume was 1.74 ml, containing 20 µl of the concentrated ethyl acetate extract as the sample, 1.6 ml of
0.1 M potassium phosphate (pH 7.6), 100 µl of
2H2O, used as the deuterium lock, and 20 µl
of 8.4 mM 4-fluorobenzoate added as an internal standard.
Concentrations of the various metabolites were calculated by comparison
of the integrals of the 19F NMR resonances of the
metabolites to the integral of the 4-fluorobenzoate resonance. Chemical
shifts are reported relative to that for CFCl3. 19F NMR chemical shift values for the various
fluorine-containing compounds were identified using authentic reference
compounds for fluoride anions and all fluorophenols. The resonances of
the different fluorocatechol and fluoromuconate metabolites have been identified and reported previously (2, 14). 1H
decoupling was achieved with a Waltz16 decoupling sequence.
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RESULTS |
Conversion of monofluorophenols by R. opacus 1cp.
Table 1 presents the characteristic mass
spectrum peaks of the various hydroxylated compounds identified in the
ethyl acetate extracts from the incubations of R. opacus 1cp with the different monofluorophenols.
Based on time-dependent TLC and HPLC patterns and MS analysis of
the compounds, the formation of catechol-type intermediates, resulting
from initial ortho hydroxylation of the phenols, could be
identified (Table 2). Conversion of
2-fluorophenol mainly resulted in the formation of
3-fluorocatechol. In incubations with
3-fluorophenol, formation of 4-fluorocatechol could be detected. Conversion of 4-fluorophenol by R. opacus 1cp resulted in
the formation of 4-fluorocatechol.
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TABLE 2.
Fluorophenol degradation and intermediate formation in
1-h incubations of R. opacus 1cp grown in the presence
of different (fluoro)phenol inducersa
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FIG. 1.
Aromatic hydroxylation of fluorophenols providing
possible pathways for formation of trihydroxy-fluorobenzene derivatives
formed from fluorocatechols. Solid arrows, pathways detected and
observed in the present study.
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Interestingly, unknown metabolites were detected in the incubation
mixtures of
R. opacus 1cp with each of the three
monofluorophenols.
These intermediates were only observed in
incubations with
R. opacus 1cp cells grown on phenol in
the presence of a fluorophenol
inducer (Table
2). Further studies were
conducted to identify
this new type of metabolites. These intermediates
were formed
in addition to the fluoromuconates, which are formed by the
cleavage
of fluorocatechols by intradiol dioxygenases known to be
present
in
R. opacus 1cp (
3). Based on the MS
characteristics (Table
1) and the exact mass of 144.0223, which is
consistent with a
C
6H
5O
3F
composition, these compounds were identified as trihydroxyfluorobenzene
derivatives.
Figure
1 presents the possible trihydroxyfluorobenzene derivatives that
may be formed from the different fluorocatechols.
It is important to
note that formation of 1,2,4-trihydroxybenzene-type
intermediates would
require the action of an aromatic
para-hydroxylase,
different from the
ortho-hydroxylating phenol
hydroxylase converting
the fluorophenols to their corresponding
catechols. It is possible
that 1,2,3-trihydroxyfluorobenzenes
(fluoropyrogallols) are formed
from fluorocatechols by the action
of the same
ortho-hydroxylating
phenol hydroxylase that
transforms fluorophenols to fluorocatechols.
This formation of
1,2,3-trihydroxyhalobenzenes in the biotransformation
of fluorophenols
has not been reported before in
prokaryotes.
Identification of the substituent pattern in the trihydroxylated
fluorobenzene compounds.
In order to establish the nature of the
trihydroxyfluorobenzene derivatives in more detail, the ethyl acetate
extracts of the incubations with 2-fluorophenol and 3-fluorophenol,
each containing one of the two different trihydroxyfluorobenzene
derivatives, were analyzed by 19F NMR.
Figure
2a presents the
19F
NMR spectrum of the ethyl acetate extract of the mixture containing
R. opacus 1cp incubated for 2
h with 2-fluorophenol.
From this spectrum it can be concluded
that the parent 2-fluorophenol
(at
141.9 ppm) has disappeared
and 3-fluorocatechol is the major
fluorine-containing compound
present. This is completely consistent
with what could be derived
from the TLC patterns at this time of
the incubation. The
19F NMR spectrum reveals the presence
of a small amount of 2-fluoromuconate
resulting from
ortho cleavage of 3-fluorocatechol. In addition,
a
19F NMR signal at
149.2 ppm is observed, probably
representing
the trihydroxyfluorobenzene metabolite detected by TLC and
MS
(Table
1).
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FIG. 2.
19F NMR spectra of ethyl acetate extracts of
2-h incubations of R. opacus 1cp with 2-fluorophenol (a) and
3-fluorophenol (b). IS, resonance from the internal standard
4-fluorobenzoate; arrows, predicted resonance positions of possible
hydroxylated catechol and hydroxyhydroquinone metabolites that can be
formed from 3-fluorocatechol (a) and 3-fluoro- and 4-fluorocatechol
(b), the initial hydroxylated products formed from these phenols.
Insets, 1H-coupled 19F NMR splitting patterns
of the peaks of the trihydroxyfluorobenzene metabolites, as well as of
the peak tentatively identified as 2-pyrone-4-fluoro-6-carboxylic acid.
For further details see text and Table 1.
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The exact substituent pattern of the trihydroxyfluorobenzene was
determined based on the chemical shift and the proton-coupled
19F NMR spectrum of this compound. In theory, the formation
of three
different trihydroxyfluorobenzene isomers can be foreseen,
taking
into account formation of 3-fluorocatechol as the major
initial
step in 2-fluorophenol conversion (Fig.
1). Two of these
metabolites,
1,2,4-trihydroxy-3-fluorobenzene and
1,2,4-trihydroxy-6-fluorobenzene,
are hydroxyhydroquinone-type
metabolites, whereas the other, 1,2,3-trihydroxy-4-fluorobenzene,
is an
ortho-hydroxylated catechol-type
metabolite.
Based on the literature (
14) it can be concluded that
incorporation of a hydroxyl moiety
ortho,
meta,
or
para with respect
to a fluorine in a benzene derivative
will result in a change
in the chemical shift of that fluorine
substituent by
23.1 ±
0.3 ppm, +1.3 ± 0.4 ppm, and
11.2 ± 0.7 ppm, respectively. The
chemical shift value of
3-fluorocatechol, which has been identified
as
140.4 ppm
(
14), and that of 4-fluorocatechol, identified
as
126.7
ppm (
14), can be used to calculate the chemical shift
values
expected for the three possible trihydroxyfluorobenzenes.
Figure
2a
indicates these chemical shift positions. This analysis
reveals that
formation of 1,2,4-trihydroxy-6-fluorobenzene (predicted
to be around
139.1 ppm and recently observed at
138.4 ppm) (
19)
and
of 1,2,4-trihydroxy-3-fluorobenzene (predicted at
163.5 and
recently
observed at
161.7) (
19) is not observed. The resonance
at
149.2 ppm, however, is in the parts per million range expected
for
1,2,3-trihydroxy-4-fluorobenzene, which is predicted to be
centered on
150.7 ppm. Final proof for the assignment of the
derivative with its
resonance at
149.2 ppm as 1,2,3-trihydroxy-4-fluorobenzene
comes from the splitting pattern observed for this signal in
proton-coupled
19F NMR measurements, revealing a
double doublet with
3J
HF = 10.0 Hz
and
4J
HF = 5.2 Hz (Fig.
2a, inset). This
confirms the presence of a
proton
ortho as well as
meta with respect to the fluorine substituent
in this
intermediate. This clearly identifies the trihydroxylated
metabolite as
1,2,3-trihydroxy-4-fluorobenzene.
Figure
2b presents the
19F NMR spectrum of the
ethyl acetate extract of the reaction mixture of
R. opacus 1cp incubated for
2 h with 3-fluorophenol. The
spectrum reveals complete transformation
of the parent substrate,
reflected by the absence of a
19F resonance peak at
116.5
ppm. Accumulation of fluorocatechols
is observed at this time of the
incubation in amounts that were
almost below the detection limit of the
19F NMR measurement (1 µM). Formation of 2-fluoro- and
3-fluoromuconate,
resulting from
ortho cleavage of
3-fluorocatechol and 4-fluorocatechol,
respectively, is also observed
in trace amounts. The main fluorine-containing
peaks detected at this
time of the incubation reflect the formation
of a large amount of
fluoride anions (at
123.0 ppm) and the formation
of unknown
intermediates with
19F chemical shift values of
90.3,
119.0,
119.1,
125.9,
141.8,
and
154.3 ppm. Based on TLC
and MS analysis (Table
1), the presence
of a trihydroxyfluorobenzene
metabolite can be
expected.
By using the chemical shift values of 3-fluorocatechol and
4-fluorocatechol (see above), chemical shift values expected for
the five possible trihydroxyfluorobenzenes that can be formed
from either 3-fluorocatechol or 4-fluorocatechol (Fig.
1)
can
be calculated. Figure
2b indicates these chemical shift
positions.
Notably, resonances corresponding to
1,2,4-trihydroxy-6-fluorobenzene
(
138.4 ppm
[
19]), 1,2,4-trihydroxy-3-fluorobenzene (
161.7
ppm
[
19]), 1,2,3-trihydroxy-4-fluorobenzene (
149.2 ppm;
see
above), and 1,2,4-trihydroxy-5-fluorobenzene (
149.8 ppm
[
19])
were not observed. However, the chemical
shift of the resonance
at
125.9 ppm corresponds to that of
1,2,3-trihydroxy-5-fluorobenzene,
which is predicted to be
125.4 ppm.
The proton-coupled splitting
pattern of the
19F NMR
resonance at
125.9 shows a clear triplet with two
3J
HF values of 10.1 Hz each (Fig.
2b, inset).
This unequivocally
shows the presence of two protons, one at each
position
ortho with respect to the fluorine substituent.
Together this identifies
the trihydroxylated fluorobenzene derivative
as 1,2,3-trihydroxy-5-fluorobenzene.
Figure
2b also presents the
1H-coupled
19F NMR
splitting pattern of the resonance at
90.3 ppm, the other major
unidentified
metabolite formed. Identification of this metabolite may
give
a clue to the possible conversion of
1,2,3-trihydroxy-5-fluorobenzene
by ring-cleaving dioxygenases. The
cleavage of 1,2,3-trihydroxybenzene
by both intra- and extradiol
dioxygenases has been described as
resulting in the formation of either
a 3-hydroxymuconate or a
2-pyrone-6-carboxylate or both
(
17). By analogy, conversion
of
1,2,3-trihydroxy-5-fluorobenzene may result in the formation
of
2-hydroxy-4-fluoromuconate or, alternatively,
2-pyrone-4-fluoro-6-carboxylate
(Fig.
3).
The
1H-coupled
19F NMR splitting pattern of the
resonance at
90.3 ppm clearly
shows a triplet with two coupling
constants of 7.9 Hz (Fig.
2b,
inset). The size of these coupling
constants is consistent with
3J
HF values
generally observed in planar aromatic geometries and
in fluorinated
pyrones (
5,
22) but is not consistent with
3J
HF values previously reported for
fluoromuconates, which vary
between 10 and 40 ppm (
2,
22).
This demonstrates that this
resonance is not indicative of
2-hydroxy-4-fluoromuconate. In
contrast, the two J
HF
coupling constants of 7.9 Hz are consistent
with what would be
expected for the two
3J
HF coupling constants in
2-pyrone-4-fluoro-6-carboxylate. The
reported chemical shifts of
19F for substituted 4-fluoropyrones are
85.7 and
86.5
ppm, while
those of the C-4-fluoro substituent of 3,4-difluoropyrones
are
between
118 and
123 ppm (
5,
22). Taking into account
a
correction for the approximately
25-ppm change in chemical shift
value upon introduction of an aromatic
ortho-fluorine
substituent
(
14,
16), the chemical shift of
2-pyrone-4-fluoro-6-carboxylate
is expected to be between
85 and
98 ppm, consistent with the
observed value of
90.3 ppm. Based
on this observation, the TLC
extracts of the incubations of
R. opacus 1cp with 3-fluorophenol
and 4-fluorophenol were
analyzed for the presence of 2-pyrone-4-fluoro-6-carboxylate.
Although the compound could not be completely purified from the
TLC
plates, a fraction containing a compound with a molecular
ion
m/z in the mass spectrum at 158 could be observed,
eliminating
a COOH fragment to give a fragment peak at
m/z 113, followed by
CO elimination to give a fragment
peak at
m/z 85. This fragmentation
pattern together
with the
19F NMR data tentatively indicates the formation
of 2-pyrone-4-fluoro-6-carboxylate
in incubations of
R. opacus 1cp with 3-fluorophenol and
4-fluorophenol
and suggests the conversion of
1,2,3-trihydroxy-5-fluorobenzene
to 2-pyrone-4-fluoro-6-carboxylate.
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FIG. 3.
Possible oxygenolytic cleavage of
1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) in analogy to
what has been observed for the conversion of 1,2,3-trihydroxybenzene by
different intradiol and extradiol dioxygenases (17). Solid
arrow, pathway proposed in the present study.
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Furthermore, the
19F NMR spectra presented in Fig.
2 also
give quantitative information. The conversion of 2-fluorophenol results
in the formation of small amounts of fluoride anions and
2-fluoromuconate
and 1,2,3-trihydroxy-4-fluorobenzene, which amount to,
respectively,
6.4, 4.6, and 5.9% of the total amount of
fluorine-containing
metabolites. This implies that formation of
4-fluoropyrogallol
efficiently competes with formation of
2-fluoromuconate from 3-fluorocatechol.
In the incubations with 3-fluorophenol, 1,2,3-hydroxy-5-fluorobenzene
and the peak tentatively assigned to 2-pyrone-4-fluoro-6-carboxylate
make up 9.7 and 9.2%, respectively, of the total amount of
fluorine-containing
intermediates detected. Eventually in all
incubations the trihydroxylated
derivatives as well as the
2-pyrone-4-fluoro-6-carboxylate disappear
from the medium, indicating
their further degradation, which,
for 1,2,3-trihydroxy-5-fluorobenzene
in particular, seems to be
accompanied by efficient defluorination,
reflected by a large
percentage of fluoride anions in the incubation
medium amounting
to 54.0% of all fluorine-containing metabolites in
the
19F NMR spectrum of Fig.
2b.
Finally, Fig.
4 presents the time course
for biotransformation of 4-fluorophenol by whole cells of
R. opacus 1cp coinduced
with 4-fluorophenol. Degradation of
4-fluorophenol and transient
formation of 4-fluorocatechol and
5-fluoropyrogallol can be observed.
After 4 to 6 h of incubation,
4-fluorophenol, 4-fluorocatechol,
and 5-fluoropyrogallol disappeared
from the reaction mixture.
Similar results were obtained for the other
two isomers (Table
2). The data in Table
2 indicate that 2-fluorophenol
and 3-fluorophenol,
when incubated with cells coinduced with the
corresponding monofluorophenol
or with 4-fluorophenol, were converted
at lower rates than 4-fluorophenol.
This indicates that, at least in
the 4-fluorophenol-induced cells,
their lower rates of conversion are
due to a lower rate of conversion
by the phenol hydroxylase
present. Table
2 also presents data
on the degradation of
4-fluorophenol by cells from
R. opacus 1cp
induced solely
with phenol. Degradation of 4-fluorophenol by these
cells is
significantly slower, suggesting the induction of phenol
hydroxylase
activity by 4-fluorophenol. Moreover, the fact that
in the incubations
with phenol-induced cells, no formation of
fluoropyrogallols was
observed suggests that the phenol hydroxylase
induced by 4-fluorophenol
is different from the one induced upon
induction by phenol.
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FIG. 4.
Time course of 4-fluorophenol degradation by whole cells
of R. opacus 1cp coinduced with 4-fluorophenol as determined
by HPLC.  , 4-fluorophenol;  , 4-fluorocatechol;  ,
5-fluoropyrogallol.
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DISCUSSION |
In the present study, the transformation of the isomeric
monofluorophenols by whole cells of R. opacus 1cp was
investigated, with particular emphasis on the nature of the
hydroxylated intermediates formed. The results obtained indicate that,
upon conversion of the fluorophenols, transient formation of
trihydroxyfluorobenzene metabolites occurs. The chemical shifts and
splitting patterns of the 19F NMR resonances in
1H-coupled 19F NMR measurements were used to
establish that 1,2,3-trihydroxyfluorobenzenes (fluoropyrogallols) occur
as new intermediates in the transformation of fluorophenols by
R. opacus 1cp.
The formation of the 1,2,3-trihydroxyfluorobenzenes from the
fluorophenols could result from two successive ortho
hydroxylation steps by the ortho-phenol hydroxylase of
R. opacus 1cp. Interestingly, a sequential ortho
hydroxylation of fluorinated substrates was recently also reported for
para-hydroxybenzoate hydroxylase from Pseudomonas
fluorescens (20). Fluoropyrogallol accumulation was
only observed in incubations with cells grown on phenol in the presence
of a fluorophenol inducer. This suggests that the phenol hydroxylase
responsible for formation of the fluoropyrogallols is different from
the phenol hydroxylase responsible for conversion of phenol in cells
grown on phenol without a fluorophenol inducer. Further
characterization of the phenol hydroxylases was hampered by their high
lability. This is a common phenomenon among Rhodococcus species, and therefore these enzymes are difficult to detect and/or purify from cell extracts (4, 11, 18, 23). Possible reasons for these difficulties in studying phenol hydroxylase activities from
Rhodococcus strains are (i) that the enzymes in question require special in vitro additions to remain in an active status or (ii) that the enzymes are of a multicomponent nature (15) and readily dissociate and/or contain one or more components which rapidly inactivate (11, 18, 23).
After several hours of incubation all fluorocatechols and
fluoropyrogallols disappeared from the reaction mixtures. This suggests that ring-cleaving enzymes may be capable of converting not only the fluorocatechols but also their ortho-hydroxylated
derivatives. Additional results of the present study tentatively
indicated the formation of 2-pyrone-4-fluoro-6-carboxylate from
1,2,3-trihydroxy-5-fluorobenzene in incubations with R. opacus 1cp. This would imply that the dioxygenase(s) present
in this species would behave similarly to
protocatechuate-3,4-dioxygenase from Pseudomonas
aeruginosa, preferentially catalyzing the conversion of
the 1,2,3-trihydroxybenzene derivative to a 2-pyrone-6-carboxylate derivative instead of to a 2-hydroxymuconate (17).
This also discriminates the dioxygenase(s) from R. opacus 1cp from the extradiol dioxygenase from P. arvilla (ATCC 23973), which catalyzes preferential ring
cleavage of 1,2,3-trihydroxybenzene to give 2-hydroxymuconate, and from the intradiol catechol dioxygenase from P. arvilla
C1 (ATCC 23974), which catalyzes the formation of a mixture of about equimolar amounts of both products (17). The isolation and
characterization of the enzyme from R. opacus 1cp that
transforms the 1,2,3-trihydroxyhalobenzene derivatives are under way.
In analogy to the experiments reported in the present study for
fluorophenols, preliminary studies using chlorophenols
provided evidence for formation of chloropyrogallols. Especially
for 2-chlorophenol and 2,3-dichlorophenol, formation of the
corresponding chloro- and dichloropyrogallol was observed in TLC and MS
experiments. For 3-chloro- and 4-chlorophenol, no accumulation of
pyrogallol metabolites occurred (Finkelstein et al., unpublished
results). These data support the link between the two types of
halophenols. Formation of 1,2,3-trihydroxychlorobenzene has been
reported in the transformation of isomeric monochlorophenols by
Penicillium simplicissimum SK9117 (13), but the
present study demonstrates for the first time the formation of this
type of hydroxylated metabolites in the transformation of halophenols
by a prokaryote.
| |
ACKNOWLEDGMENTS |
This work was supported by EC grant ERB IC15-CT96-0103, the EU
large-scale WNMRC facility (grant ERBFMGECT 950066), and grant 047.007.021 for Dutch-Russian research cooperation from the Dutch Scientific Organisation NWO.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biomolecular Sciences, Laboratory of Biochemistry, Wageningen
University, Dreijenlaan 3, NL-6703 HA Wageningen, The Netherlands.
Phone: 31-317-482868. Fax: 31-317-484801. E-mail:
ivonne.rietjens{at}P450.BC.WAU.NL.
| |
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Applied and Environmental Microbiology, May 2000, p. 2148-2153, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
