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J. Biol. Chem., Vol. 275, Issue 29, 21889-21895, July 21, 2000
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From
Received for publication, March 6, 2000, and in revised form, April 13, 2000
The enzymes benzoyl-CoA reductase and
cyclohex-1,5-diene-1-carbonyl-CoA hydratase catalyzing the first steps
of benzoyl-CoA conversion under anoxic conditions were purified from
the denitrifying bacterium, Thauera aromatica. Reaction
products obtained with [ring-13C6]benzoyl-CoA and
[ring-14C]benzoyl-CoA as substrates were
analyzed by high pressure liquid chromatography and by NMR
spectroscopy. The main product obtained with titanium(III) citrate or
with reduced [8Fe-8S]-ferredoxin from T. aromatica as
electron donors was identified as cyclohexa-1,5-diene-1-carbonyl-CoA. The cyclic diene was converted into
6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures
containing reductase, hydratase, and sodium dithionite or a mixture of
sulfite and titanium(III) citrate as reducing agent afforded
cyclohex-2-ene-1-carbonyl-CoA and
6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the
first electron transfer to the model compound
S-ethyl-thiobenzoate yielding a radical anion was
determined by cyclic voltammetry as Traditionally, the biodegradation of aromatic compounds has been
considered as a domain of aerobic metabolism. More recently, an
increasing number of bacteria has been shown to metabolize aromatic
compounds under anoxic conditions. Benzoyl-CoA (Fig. 1, compound 1) has been
identified as a central intermediate in anoxic degradation of many
aromatic substrates (for review see Refs. 1-4).
Benzoyl-CoA reductase has been purified from the denitrifying bacterium
Thauera aromatica (5). The 160-kDa protein consists of four
different subunits specified by the bcrCBAD genes. The enzyme contains a minimum of two Fe-S clusters (5, 6) and is only
active under strictly anoxic conditions (5). The reduction of
benzoyl-CoA is coupled to the hydrolysis of two molecules of ATP/molecule of benzoyl-CoA. The natural electron donor for the enzyme-catalyzed reduction of benzoyl-CoA is a [8Fe-8S]-ferredoxin with a midpoint potential of In analogy to the Birch reduction of aromatic compounds by metallic
sodium in liquid ammonia, the enzyme-catalyzed reduction of benzoyl-CoA
has been proposed to proceed by two one-electron transfer reactions,
each of which is followed by a protonation step (8, 9). The crucial
step is the first electron transfer to the aromatic ring yielding a
radical anion. EPR studies indicated that the hydrolysis of ATP is
responsible for conformational changes in the vicinity of [4Fe-4S]
clusters, most probably resulting in a lowered reduction potential of
the clusters (6).
[ring-13C6]Cyclohexa-1,5-diene-1-carbonyl-CoA
was identified in a product mixture obtained after incubation of a
crude extract preparation from T. aromatica containing
[ring-13C6]benzoate, coenzyme A,
ATP, Mg2+, and titanium(III) citrate (8).
Cyclohexa-1,5-diene-1-carbonyl-CoA (Fig. 1, compound
2) was proposed tentatively to be the committed product of
benzoyl-CoA reductase. Furthermore, an enzyme (Dch protein;
subsequently designated as 1,5-dienoyl-CoA hydratase) catalyzing the
formation of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (compound
3) by addition of water to the cyclic 1,5-diene (compound 2) has been purified and characterized
(10).
In contrast to the results obtained with T. aromatica,
cyclohexa-2,5-diene-1-carboxylate (Fig.
2, compound 4) and
cyclohexa-1,4-diene-1-carboxylate (compound 5) were
reported as products of benzoic acid reduction in cell suspensions of
the phototrophic bacterium Rhodopseudomonas palustris. The
structure assignments were based on gas chromatography and mass
spectrometry after hydrolytic treatment of the reaction mixture
(11).
Nonaromatic Products from Anoxic Conversion of Benzoyl-CoA
with Benzoyl-CoA Reductase and Cyclohexa-1,5-diene-1-carbonyl-CoA
Hydratase*
,,,
Mikrobiologie, Institut für Biologie II,
Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg,
Germany, the § Institut für Organische Chemie und
Biochemie, Technische Universität München,
Lichtenbergstrasse 4, D-85747 Garching, Germany, and the
¶ Institut für Physikalische Chemie, Albertstrasse 21, D-79104 Freiburg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.9 V versus a
standard hydrogen electrode. The energetics of enzymatic ring reduction
of benzoyl-CoA are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
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Fig. 1.
Postulated initial reactions of the
benzoyl-CoA pathway in the denitrifying bacterium T. aromatica catalyzed by benzoyl-CoA reductase (BrcCBAD
protein) and 1,5-dienoyl-CoA hydratase (Dch protein) (5, 6,
10).
450 mV (versus a standard
hydrogen electrode) (7). Alternatively, titanium(III) citrate,
dithionite, or reduced methyl viologen can serve as artificial
reductants. Some CoA-thioesters of fluoro-, amino-, and hydroxybenzoate
can be reduced by benzoyl-CoA reductase albeit at low rates. Moreover, low molecular mass compounds such as hydroxylamine and azide are also
reduced. All these reactions strictly depend on the hydrolysis of ATP
(5).
View larger version (22K):
[in a new window]
Fig. 2.
Products of benzoate reduction in the
phototrophic bacterium R. palustris (11).
The present study was initiated to establish the product of benzoyl-CoA
reductase from T. aromatica. For this purpose, benzoyl-CoA labeled with 13C or 14C was incubated with
purified benzoyl-CoA reductase using various electron donors. Enyzme
products were analyzed by
HPLC1 and two-dimensional
homonuclear and heteronuclear NMR spectroscopy. Furthermore, the effect
of a thioester residue on the reduction potential of the aromatic
moiety was determined by cyclic voltammetry using
S-ethyl-thiobenzoate as a model compound.
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EXPERIMENTAL PROCEDURES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials-- Reagents were obtained from Aldrich, Fluka, Merck, Sigma, Roth, Roche Molecular Biochemicals, and Gerbu. Chromatography materials were obtained from Amersham Pharmacia Biotech, Bio-Rad, and Merck, respectively. [ring-13C6]Benzoic acid was purchased from MS Isotopes, and [ring-14C]benzoate was obtained from American Radiolabeled Chemicals Inc. and Biotrend Chemikalien GmbH.
Bacterial Strains-- T. aromatica strain K172 (DSM 6984) was isolated in our laboratory (12, 13) and has been deposited in Deutsche Sammlung von Mikroorganismen (Braunschweig, Germany). Clostridium pasteurianum (ATCC 6013) was obtained from the American Type Culture Collection.
Synthesis of CoA
Thioesters--
[ring-13C6]Benzoyl-CoA
was obtained via esterification of benzoic acid with
N-hydroxysuccinimide (14).
[ring-14C]Benzoyl-CoA and
cyclohex-1-ene-1-carbonyl-CoA were prepared enzymatically from
[ring-14C]benzoate (specific radioactivity,
2.025 GBq mmol
1) and cyclohex-1-ene-1-carboxylate,
respectively, using coenzyme A and enriched benzoate-CoA ligase from
T. aromatica as described earlier (15).
[ring-13C6]benzoyl-CoA and
[ring-14C]benzoyl-CoA were mixed (600 kBq
mg1) and co-purified by semi-preparative HPLC as
described (10). The yield was 50-80%.
Synthesis of S-Ethyl-thiobenzoate--
Treatment of benzoic acid
phenyl ester with sodium ethyl mercaptide afforded
S-ethyl-thiobenzoate (16). An aliquot (80 µl) of the
reaction mixture was dissolved in 920 µl of toluene and purified by
preparative thin layer chromatography using silica gel 60 plates
(Merck, 2 mm, 20 × 20 cm) with toluene as solvent. A strip of the
plate was sprayed with 0.01% fluorescein (m/v) in ethanol, and the
bands were visualized under ultraviolet light (254 nm). The product
band was extracted with toluene and dried under reduced pressure
(1H NMR using CDCl3 as solvent: 
(ppm); 1.37 (3H, t, CH3), 3.10 (2H, q, CH2),
7.33-8.00 (5H, m, C6H5).
Growth of Bacterial Cells and Preparation of Cell Extract-- T. aromatica was grown anoxically at 28 °C in a mineral salt medium containing a mixture of 4-hydroxybenzoate and nitrate (ratio 1:3.5) as sole source of carbon and energy (12). Hydroxybenzoate and nitrate were continuously fed at a molar ratio of 1:3.5. Cells were harvested by centrifugation under anaerobic conditions and were stored in liquid nitrogen. Cell extract was prepared as described (17).
Purification of Benzoyl-CoA Reductase--
Benzoyl-CoA reductase
was partially purified from T. aromatica (wet cell mass,
200 g) under strictly anaerobic conditions in a glove box
containing an atmosphere of N2/H2 (95:5, v/v)
as described earlier (5). Benzoyl-CoA reductase was further purified by
chromatography on a column of Blue Sepharose CL 6B (Amersham Pharmacia
Biotech, 4.2 × 12.5 cm) that had been equilibrated with 20 mM Tris-HCl, pH 7.5. The column was developed with three
bed volumes of equilibration buffer and was then developed with 20 mM Tris-HCl, pH 7.5, containing 500 mM KCl
(flow rate, 2 ml min1). Fractions were assayed for
benzoyl-CoA reductase (5) and cyclohexa-1,5-diene-1-carbonyl-CoA
hydratase activity (10).
Purification of Ferredoxin-- Ferredoxin was purified from T. aromatica (wet cell mass, 200 g) as described earlier (7). The yield was 60 mg of pure protein.
Assay for Benzoyl-CoA Reductase--
Benzoyl-CoA reductase was
assayed spectrophotometrically using reduced methyl viologen as
electron donor (5) or by HPLC as described earlier (6). The reaction
mixture for HPLC analysis contained 150 mM MOPS/KOH, pH
7.3, 10 mM MgCl2, 7.5 mM ATP, 10 mM phosphoenolpyruvate, 0.2 mM
[ring-14C]benzoyl-CoA, 80 nkat of pyruvate
kinase, and purified benzoyl-CoA reductase from T. aromatica
(0.1-0.2 unit) in a total volume of 1 ml. Titanium(III) citrate or
sodium dithionite were added to a final concentration of 5 mM. Assays using reduced ferredoxin (37 µM)
from T. aromatica as reductant were performed under an atmosphere of 100% hydrogen. Reduction of T. aromatica
ferredoxin was catalyzed by a ferredoxin-free crude preparation of
hydrogenase from C. pasteurianum (1.5 units
mg1) that had been prepared as described previously (7).
When ferredoxin was used, other reductants were omitted. Unless
otherwise stated, the reaction was started by adding 50 µl of a
purified benzoyl-CoA reductase solution.
Assay of Dienoyl-CoA Hydratase--
The enzymatic hydration of
cyclohex-1,5-diene-1-carbonyl-CoA and the reverse reaction, dehydration
of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, were determined by a
continuous spectrophotometric assay (
= 310 nm) at 37 °C as
described (10). Additionally, dienoyl-CoA hydratase was determined by
HPLC analysis of substrate consumption and product formation. The assay
mixture for HPLC analysis contained 150 mM MOPS/KOH, pH
7.3, 0.1 mM 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, and
approximately 0.1 unit of dienoyl-CoA hydratase in a volume of 0.25 ml.
The mixture was incubated at 37 °C. After 10 min, equilibrium was
reached, and approximately one-half of
6-hydroxycyclohex-1-ene-1-carbonyl CoA was converted into the cyclic
1,5-diene. Subsequently, sodium dithionite, sodium sulfite, or a
mixture of sodium sulfite and titanium(III) citrate were added to a
final concentration of 1 mM each. Aliquots were retrieved
at intervals and applied to a Lichrosphere 100 RP C-18 HPLC column
(Merck; bed volume, 1 ml; flow rate, 1 ml min1) that had
been equilibrated with 50 mM potassium phosphate, pH 6.7, containing 10% acetonitrile (v/v). The column was developed with the
equilibration buffer. The effluent was monitored by scintillation counting (Ramona, Raytest, Straubenhardt, Germany) or photometrically at 260 nm. Retention volumes were 11.1 ml for benzoyl-CoA, 13.9 ml for
cyclohexa-1,5-diene-1-carbonyl CoA, 4.4 ml for
6-hydroxycyclohex-1-ene-1-carbonyl CoA, 2.5 ml for
6-hydroxycyclohex-2-ene-1-carbonyl-CoA, and 16.5 ml for
cyclohex-2-ene-1-carbonyl-CoA. The assignment of the HPLC peaks to
individual CoA-thioesters was performed by NMR analysis of
13C-labeled compounds (see below).
Sample Preparation for NMR Analysis--
Reaction mixtures (5 ml) contained 150 mM MOPS/KOH, pH 7.3, 10 mM
MgCl2, 7.5 mM ATP, 10 mM
phosphoenol pyruvate, and 400 nkat pyruvate kinase, and 10 mM titanium(III) citrate or 10 mM sodium dithionite. The reaction was started by the addition of a mixture of
[ring-14C]benzoyl-CoA (44.4 kBq) and 5 mg of
[ring-13C6]benzoyl-CoA. The
mixtures were incubated under anoxic conditions at 37 °C for 20-40
min. The reaction was stopped by stirring for 15 min at 0 °C under
an aerobic atmosphere. The solution was lyophilized, and the powder was
dissolved in 1 ml of 20 mM ammonium formate, pH 5. The
solution was applied to a Lichrosorb RP-C18 column (25 × 2 cm;
Merck; flow rate, 8 ml min1), which had been equilibrated
with 20 mM ammonium formate, pH 5. After washing the column
with two bed volumes of 10% methanol (v/v) in the same buffer, the CoA
thioesters were eluted with 98% methanol (v/v) in the same buffer.
Methanol was evaporated under reduced pressure at 30 °C, and the
residue was lyophilized. Product recovery was determined by liquid
scintillation counting and was 60-80% of the initial amount of
14C added to the assay.
When reduced ferredoxin was used as reductant, the reaction was performed in a 100-ml stoppered bottle containing 9.6 ml of the standard assay mixture. The bottle was flushed with hydrogen for 20 min under continuous stirring. A ferredoxin-free hydrogenase preparation (H2 reduction activity: 5-6 µmol/min) from C. pasteurianum (7), [ring-13C6]benzoyl-CoA (7 mg), 85 kBq [ring-14C]benzoyl-CoA, and 3.5 mg ferredoxin from T. aromatica (final concentration, 37.5 µM) were added. The mixture was flushed with hydrogen for 20 min under continuous stirring. The assay was started by the addition of benzoyl-CoA reductase (0.8 unit) and was terminated after 15 min by adding 250 µl of 1% formic acid (final pH, 3-4). The mixture was then lyophilized.
NMR Spectroscopy-- The lyophilized samples were dissolved in D2O at pH 6 (uncorrected glass electrode reading). 1H and 13C NMR spectra were measured at 17 °C using a four-channel Bruker DRX500 spectrometer equipped with pulsed field gradient accessory, a lock switch unit, and an ASPECT station. One-dimensional and two-dimensional HMQC, HMQC-TOCSY, and INADEQUATE experiments were performed as described earlier (8). The duration of the 1H spin-lock was 60 ms in the HMQC-TOCSY experiment.
Cyclic Voltammetry-- The midpoint potential of S-ethyl-thiobenzoate was determined at 25 °C by cyclic voltammetry. All measurements were carried out in super-dry acetonitrile with tetrabutyl ammonium hexafluorophosphate as supporting electrolyte. A three-electrode cell compartment was used throughout. For the electrochemical measurements, a potentiostat model 173 and a programmer model 175 (EG&G, Burlington, Vermont) were used. All potentials were calibrated against a ferrocene/ferrocenium couple.
Miscellaneous Methods--
Polyacrylamide gel electrophoresis
and SDS-polyacrylamide gel electrophoresis were performed as described
by Schägger and von Jagow (18). Proteins were visualized by
Coomassie Blue staining (19). Protein was determined by the methods of
Bradford (20) or Lowry et al. (21) using bovine serum
albumin as standard.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Benzoyl-CoA reductase was purified from T. aromatica as
described earlier (5). The enzyme was enriched 40-fold by four chromatographic steps with a yield of 25% to a specific activity of
0.48 µmol min1 mg1 (measured with reduced
methyl viologen as electron donor (5)) and >95% purity as estimated
by SDS gel electrophoresis. This protein fraction contained benzoyl-CoA
reductase (7 µmol min1) and
cyclohexa-1,5-diene-1-carbonyl-CoA hydratase (86 µmol
min1) activity. Further purification of benzoyl-CoA
reductase was achieved by affinity chromatography on Cibachron Blue
(Amersham Pharmacia Biotech). The effluent of the column contained
benzoyl-CoA reductase activity (4.5 µmol min1) and 7%
of the initial hydratase activity (6.0 µmol min1).
Using buffer containing 0.5 M KCl, 70% of the initial
dienoyl-CoA hydratase activity could be eluted from the column.
A mixture of benzoyl-CoA reductase (7 µmol min1) and
1,5-dienoyl-CoA hydratase (86 µmol min1) was incubated
with [ring-14C]benzoyl-CoA in the presence of
reduced ferredoxin or titanium(III) citrate. Reversed phase HPLC of the
reaction mixture showed four peaks (1, 2, 3, and F) reflecting at least
three radioactive products (Fig.
3A). The compound reflecting
peak 3 was apparently more polar than benzoyl-CoA (peak 1, compound 1), product 2 was less polar than
benzoyl-CoA, and product F had the same retention volume as free
aromatic carboxylic acids and was probably formed by hydrolytic
cleavage of the thioester.
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Subsequent experiments were performed with
[ring-13C6]benzoyl-CoA as
substrate in an attempt to optimize the sensitivity and selectivity of
NMR detection. The reaction mixture was desalted and was analyzed by
NMR spectroscopy. Carbon atoms derived from the
13C-enriched phenyl ring of benzoyl-CoA substrate gave 12 intense 13C NMR signals (Fig.
4) that were attributed to two products
(compounds 2 and 3) by two-dimensional NMR
experiments. More specifically, two well separated spin systems were
gleaned from a HMQC-TOCSY experiment, establishing the connectivities
between 13C and 1H atoms as well as between
1H atoms of the 13C-labeled ring moieties (Fig.
5 and Table
I).
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Each of the 12 13C signals described above was split into a double doublet or a pseudo-triplet by 13C13C coupling constants of 30-70 Hz. Apparently, each of the observed 13C atoms was directly bonded to two adjacent 13C atoms. This result suggests that both products retained the connectivity of the ring carbon atoms of the benzoyl substrate. Some of the signals showed additional fine splittings (coupling constants of 1-8 Hz; Table I) as a result of 13C13C couplings via two or three bonds.
Four of the six intense 13C NMR signals of compound 2 had chemical shifts values (121.9, 131.6, 137.4, and 140.6 ppm; cf. Fig. 4 and Table I) consistent with sp2-hybridization of the corresponding carbon atoms. Two signals were detected at 27.7 and 22.8 ppm reflecting two sp3-hybridized carbon atoms. This 13C NMR signature in conjunction with the detected 13C13C coupling pattern was consistent with a cyclic diene structure. Two-dimensional INADEQUATE, HMQC, and HMQC-TOCSY experiments (Table I and Fig. 5) afforded the entire network of carbon-carbon, carbon-proton, and proton-proton connectivities (28 detected homo- and heterocorrelations between 1H and 13C of the carbocyclus). Specifically, HMQC spectroscopy revealed correlations of the 13C NMR signals at 121.9, 131.6, and 140.6 ppm to 1H NMR signals at 6.22, 6.02, and 6.97 ppm, respectively. The 13C signal at 137.4 ppm did not correlate to any 1H NMR signal in the HMQC experiment. This finding suggested a cyclohexadiene-1-carbonyl derivative. Carbon-carbon coupling constants (68.8 and 53.0 Hz) detected for the NMR signal at 137.4 ppm (C-1) as well as INADEQUATE spectroscopy showed that C-1 was adjacent to two sp2-hybridized carbon atoms with 13C NMR signals at 140.6 ppm (C-2) and 121.9 ppm (C-6). The signal at 121.9 ppm (C-6) gave an additional carbon-carbon correlation to the signal at 131.6 ppm (C-5), indicating a cyclohexa-1,5-diene-1-carbonyl system. The presence of a CoA residue was revealed by the 1H NMR data (not shown), thus establishing compound 2 as cyclohexa-1,5-diene-1-carbonyl-CoA.
The second product in the mixture (compound 3) was identified as 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the same approach, as described above for compound 2 (Table I). Compound 3 had been identified earlier as the reaction product of 1,5-dienoyl-CoA hydratase from T. aromatica (10). Cyclohexa-2,5-diene-1-carbonyl-CoA and cyclohexa-1,4-diene-1-carbonyl-CoA, which had been reported as products of benzoyl-CoA reduction catalyzed by benzoyl-CoA reductase in R. palustris (11), were not found in our reaction mixtures.
After prolonged incubation of
[ring-13C6]benzoyl-CoA with
benzoyl-CoA reductase, two minor products,
[ring-13C6]cyclohex-1-ene-1-carbonyl-CoA
(Fig. 6, compound 6) and [ring-13C6]2-hydroxycyclohexane-1-carbonyl-CoA
(7), were identified by NMR (data not shown). The NMR data
of these compounds have been described elsewhere (10). We suggest
tentatively that these compounds resulted from reduction of
2 by benzoyl-CoA reductase and subsequent hydration
catalyzed by cyclohexa-1,5-diene-1-carbonyl-CoA hydratase (Fig. 6). The
formation of these compounds was not observed when benzoyl-CoA
was still present in the assay mixture as shown in Fig. 3 (A
and B).
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As reported earlier, dithionite can also act as an artificial electron donor for benzoyl-CoA reductase with rates comparable with other artificial electron donors (5). Surprisingly, HPLC analysis of the products formed from incubation of [ring-14C]benzoyl-CoA with dithionite and a protein mixture containing benzoyl-CoA reductase and 1,5-dienoyl-CoA hydratase revealed a product pattern (Fig. 3B) that was different from that obtained with titanium(III) citrate or reduced ferredoxin (Fig. 3A). Peaks reflecting the cyclic 1,5-diene (compound 2) or the cyclic 6-hydroxy-1-monoene (compound 3) were not observed with dithionite as artificial electron donor. On the other hand, two novel peaks reflecting compounds 8 and 9 were detected (Fig. 3B). Moreover, when dithionite (5 mM) was added to a benzoyl-CoA reductase assay that had been started with titanium(III) citrate as electron donor, the HPLC pattern shown in Fig. 3A changed to that shown in Fig. 3C.
Compound 8 and compound 9 formed by enzymatic
conversion of
[ring-13C6]benzoyl-CoA were
analyzed by NMR spectroscopy (Table I). The detected 13C
NMR chemical shifts (120-140 ppm) and coupling signatures (double doublets or triplets) were consistent with cyclic hexamonoene motifs
for both compounds. Notably, each of the 13C atoms
correlated to directly bonded 1H atoms in the HMQC
experiment, thus indicating that the C-1 positions of both compounds
carried a hydrogen atom and were not involved in a double bond.
INADEQUATE and HMQC-TOCSY experiments afforded the entire network of
carbon-carbon and carbon-proton bonds establishing compound
9 as
[ring-13C6]cyclohex-2-ene-1-carbonyl-CoA
and compound 8 as
[ring-13C6]6-hydroxycyclohex-2-ene-1-carbonyl-CoA
(Fig. 7). No cyclic diene was detected
with dithionite as electron donor.
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To clarify the effects of electron donors on the product pattern, we
analyzed the effect of sodium dithionite on a benzoyl-CoA reductase
free preparation of 1,5-dienoyl-CoA hydratase by HPLC. Under
equilibrium conditions, similar amounts of
cyclohexa-1,5-diene-1-carbonyl CoA (peak 2) and
6-hydroxycyclohex-1-ene-1-carbonyl-CoA (peak 3) were present
in assays of the hydratase (Fig.
8A). After addition of sodium
dithionite, both compounds were converted into
6-hydroxycyclohex-2-ene-1-carbonyl-CoA (Fig. 8, B and
C, peaks 8). Fig. 8B shows that an
additional intermediate apolar product (reflecting peak 10) is formed
after 1 min of incubation. After 10 min of incubation this peak
disappeared in the chromatogram (Fig. 8C). It is conceivable
that peak 10 represents cyclohexa-2,5-diene-1-carbonyl CoA (Fig. 7,
compound 10), which can be converted rapidly into
compound 8 by the addition of water. When sodium dithionite was replaced by 1 mM sodium sulfite, the formation of
compound 8 could not be detected, whereas a mixture
containing 1 mM sodium sulfite and 1 mM
titanium(III) citrate had the same effect as sodium dithionite (not
shown). In the absence of 1,5-dienoyl-CoA hydratase, no spontaneous
reaction of dithionite with 2 and 3 could be
observed.
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For the energetics of benzoyl-CoA reduction, the transfer of the first electron to the aromatic ring of benzoyl-CoA yielding a radical anion is considered as the crucial step in the reaction catalyzed by benzoyl-CoA reductase (9). A highly negative redox potential is expected for this reaction. The natural substrate benzoyl-CoA is not suitable for the direct determination of this potential because irreversible follow-up reactions occur in protic solvents. Therefore, the substrate analog S-ethyl-thiobenzoate, which is soluble in aprotic solvents, was synthesized and analyzed by cyclic voltammetry.
The thioester was reduced at a midpoint potential of 2.1 V
versus an Ag/AgCl reference electrode. The reaction was
reversible; minor waves appeared in the reverse scan at a potential of
approximately 0.5 V versus Ag/AgCl indicating, a second
slow process (data not shown). This follow-up product probably results
from protonation of the radical anion yielding a free radical. Residual
traces of water in the solvent could be the proton donor for such species.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The structures of six CoA thioesters obtained after incubation of [ring-13C6]benzoyl-CoA with purified benzoyl-CoA reductase (BcrCBAD protein) and 1,5-dienoyl CoA hydratase (Dch protein) from T. aromatica were established by NMR analysis. The use of multiply 13C labeled substrate facilitated signal assignments by two-dimensional INADEQUATE, HMQC, and HMQC-TOCSY spectroscopy and enabled NMR analysis with product mixtures.
Specifically, the NMR data established cyclohexa-1,5-diene-1-carbonyl-CoA (compound 2) as the product of benzoyl-CoA reduction with titanium(III) or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors. This is in agreement with the results of Koch et al. (8), who identified this compound as an early product in experiments with crude cell extracts of T. aromatica. The formation of this cyclic hexadiene implicates a two-electron reduction of benzoyl-CoA and fits the previously determined stoichiometry (6). The identification of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (compound 3) as next intermediate in the benzoyl-CoA pathway in T. aromatica further supports the cyclic 1,5-diene structure, because the 6-hydroxy-1-ene can be formed by the enzymatic addition of water to the conjugated double bonds of the diene (10), either via a 1,4- or via a 1,2-addition mechanism.
The four genes of benzoyl-CoA reductase from T. aromatica and R. palustris show high similarity (78% sequence identity) (22, 23). Interestingly, Gibson and Gibson (11) detected cyclohexa-2,5-diene-1-carboxylate (compound 4) and cyclohexa-1,4-diene-1-carboxylate (compound 5) as products of benzoate reduction in cell suspension experiments with R. palustris. These compounds were identified by gas chromatography/mass spectrometry techniques after derivatization of the free acids to the corresponding methyl esters. The discrepancy to our results might have different reasons. Either benzoyl-CoA reduction proceeds via different mechanisms in R. palustris as discussed elsewhere (3, 22), or the diene isomers detected by GC/MS were artificially formed during derivatization of the products.
In the so-called Birch reduction of benzoic acid with elemental sodium or lithium as reductants, 2,5-cyclohexadiene-1-carboxylate (compound 4) is formed under kinetic control (24). The electron withdrawing character of the acid group of benzoate stabilizes the anion radical at carbon 1 of the ring; the second electron adds in para position because of electrostatic repulsion. However, in the presence of a strong base, the thermodynamically more stable conjugated 1,5-diene can be obtained (25, 26). This base-catalyzed rearrangement seems to occur in the catalytic cycle of T. aromatica.
The formation of the hex-1-ene compound (compound 6) at a slow rate (5) explained by a two-electron reduction of the 1,5-diene (compound 2) catalyzed by benzoyl-CoA reductase. Although the hex-1-ene (compound 6) and the 2-hydroxyhexane compound (compound 7) may play an important role in the aromatic metabolism of R. palustris (3, 22), their formation in T. aromatica appears as an in vitro effect without metabolic relevance.
The natural electron donor, reduced ferredoxin, and the artificial reductant titanium(III) citrate yielded the same products. Surprisingly, cyclohex-2-ene-1-carbonyl-CoA (compound 9) and 6-hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) were identified as products of benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase when dithionite was used as an artificial electron donor. Our results strongly suggest that the artificial formation of the 6-hydroxy-2-ene (compound 8) was a product of dienoyl-CoA hydratase activity via a set of artificial isomerization and hydration reactions. In this case, cyclohexa-2,5-diene-1-carbonyl-CoA (compound 10) is suggested as an intermediate isomerization product (Fig. 7). Under anaerobic conditions in the presence of benzoyl-CoA reductase, MgATP, and an excess of dithionite, this hypothetical diene is further reduced to cyclohexa-2-ene-1-carbonyl-CoA (compound 9). The following observations support our suggestions: (i) When sodium dithionite was added to a benzoyl-CoA reductase/1,5-dienoyl-CoA hydratase assay that had been started with Ti(III) citrate as electron donor and benzoyl-CoA as substrate, the product pattern shifted immediately from the cyclohexa-1,5-diene/6-hydroxymonene mixture (reflecting peaks 2 and 3 in the HPLC chromatogram; cf. Fig. 8A) to a cylohexa-2-ene/6-hydroxy-2-ene mixture (reflecting peaks 9 and 8 in the HPLC chromatogram; cf. Fig. 8C). (ii) Under aerobic conditions, when the hydratase was fully active and benzoyl-CoA reductase was irreversibly inactivated, addition of dithionite induced the formation of 6-hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) as the unique product from a cyclohexa-1,5-diene/6-hydroxymonene mixture. 6-Hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) is considered to be a dead end product that is not in equilibrium with the corresponding diene 10 (Fig. 7). A mixture of sulfite and Ti(III) citrate could replace dithionite in the artificial product formation, whereas sulfite alone had no effect on dienoyl-CoA hydratase activity. We assume that a sulfoxide radical anion is formed from sulfite by reduction with Ti(III) citrate as an active species; it is known that anaerobic dithionite solutions contain the same radical anion (27).
For energetics of anaerobic enzymatic ring reduction, a Birch mechanism
has been proposed for enzymatic reduction of the aromatic ring, which
predicts alternating one-electron transfer and protonation steps (8,
9). A ketyl radical anion at the carbonyl C atom of the thioester group
rather than a benzene radical anion was suggested as the first
intermediate. In case of benzene reduction, the potential for the first
electron transfer step is 3.1 V (versus a normal hydrogen
electrode) (28). A thioester group at the aromatic ring should lower
this potential significantly (9). Indeed, using
S-ethyl-thiobenzoate the potential is lowered to 1.9 V
(versus a normal hydrogen electrode). This drastic effect illustrates the importance of the thioester group in enzymatic reduction of benzoyl-CoA.
However, the energetics of enzymatic ring reduction are still not
clear. The two electron reduction of the aromatic ring is driven by the
stoichiometric hydrolysis of 2 ATP to 2 ADP + 2 Pi. If the
free hydrolysis energy of both ATP molecules (~100 kJ) is coupled
to a single electron transfer the redox potential can maximally be
lowered by 1 V. Assuming this situation, the potential of the first
electron transferred from the natural electron donor ferredoxin with
E'° = 0.45 (versus normal hydrogen electrode) could be lowered to 1.45 V in enzymatic aromatic ring reduction. In
conjunction with the determined potential of the model compound S-ethyl-thiobenzoate (1.9 V), the hydrolysis of ATP
appears to be insufficient to enable ring reduction. However, partial
protonation of the electron accepting carbonyl function by a protonated
amino acid residue or a protonated Fe-S cluster could result in a more positive redox potential for the first electron transfer yielding the
ketyl radical anions 11 and 12 (Fig.
9) as proposed by Buckel and Keese (9).
The subsequent protonation of the radical anion seems to be highly
exergonic, thus driving the endergonic first electron transfer step.
The transfer of the second electron to the protonated free radical
(compound 13) should require less energy than the formation
of the radical anion, and protonation of compound 14 to the
diene product (compound 2) is considered a highly exergonic
reaction.
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ACKNOWLEDGEMENTS |
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We thank A. Werner and F. Wendling for expert help with the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Inst. für
Biologie II, Mikrobiologie, Schänzlestr. 1, D-79104 Freiburg,
Germany. Tel.: 49-761-2032649; Fax: 49-761-2032626; E-mail:
fuchsgeo@uni-freiburg.de.
Published, JBC Papers in Press, April 13, 2000, DOI 10.1074/jbc.M001833200
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; HMQC, heteronuclear multiple quantum correlation; TOCSY, total correlation spectroscopy; INADEQUATE, incredible natural abundance double quantum transfer spectroscopy; Ti(III), titanium(III).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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