From the Department of Civil and Environmental
Engineering, Environmental Engineering and Science, Stanford
University, Stanford, California 94305, the § Department of
Biochemistry, Swammerdam Institute for Life Sciences, University of
Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The
Netherlands, and the
Department of Biological Sciences, Stanford
University, Stanford, California 94305
Received for publication, October 17, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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The novel enzyme benzylsuccinate synthase
initiates anaerobic toluene metabolism by catalyzing the addition of
toluene to fumarate, forming benzylsuccinate. Based primarily on its
sequence similarity to the glycyl radical enzymes, pyruvate
formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate
synthase was speculated to be a glycyl radical enzyme. In this report
we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium
Azoarcus sp. strain T harbors an oxygen-sensitive stable
organic free radical. The EPR signal of the radical was centered
at g = 2.0021 and was characterized by a major 2-fold splitting of
about 1.5 millitesla. The strong similarities between the EPR signal of
the benzylsuccinate synthase radical and that of the glycyl radicals of
pyruvate formate-lyase and anaerobic ribonucleotide reductase provide
evidence that the benzylsuccinate synthase radical is located on a
glycine residue, presumably glycine 828 in Azoarcus sp.
strain T benzylsuccinate synthase.
Benzylsuccinate synthase initiates anaerobic toluene
mineralization in denitrifying bacteria by catalyzing the addition of toluene to fumarate, forming benzylsuccinate (Fig.
1; Refs. 1 and 2). This toluene fumarate
addition reaction may be a general mode for anaerobic toluene
metabolism as it has been demonstrated recently in phylogenetically
distant bacteria, including several toluene-mineralizing, denitrifying
(1, 2), and sulfate-reducing (3, 4) bacteria, as well as a
toluene-utilizing phototrophic bacterium (5). Furthermore, this type of
fumarate addition reaction may be a general strategy for activating
methylbenzenes in the absence of molecular oxygen as it has been shown
to be the initial step in anaerobic m-xylene mineralization
(6) and has been implicated as the initial step in anaerobic
m-cresol mineralization (7). Unlike the initial activation
steps in aerobic methylbenzene metabolism whereby the methylbenzene is oxidized directly by molecular oxygen as a cosubstrate (8, 9),
benzylsuccinate synthase catalyzes a strictly anaerobic, nonredox
reaction. Notably, the benzylsuccinate synthase reaction also seems to
be a novel biochemical means for forming a new carbon-carbon bond.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The benzylsuccinate synthase
reaction.
Benzylsuccinate synthase, which has been characterized at both the
biochemical and molecular level
(10-12),1 is believed to be
a heterohexamer (2
2
2)
composed of three subunits with molecular masses of approximately 98 (
subunit), 8.6 (
subunit), and 6.6 (
subunit) kDa. The
predicted amino acid sequence of the
subunit of benzylsuccinate
synthase (11, 12)1 has strong homology to the well
characterized glycyl-radical enzymes, pyruvate formate-lyase
(PFL)2 and anaerobic
ribonucleotide reductase (ARNR), including a conserved glycine residue
and a conserved cysteine residue. Although all of the reported PFLs
have two conserved vicinal cysteines, cysteine 418 and cysteine 419 in
Escherichia coli PFL, benzylsuccinate synthases and ARNRs
have only one conserved cysteine, corresponding to cysteine 419 of
E. coli PFL. The conserved amino acids in PFL (13-16) and ARNR (17, 18) are essential for catalysis of their respective reactions. The catalytically active forms of PFL (13-16, 19, 20) and ARNR (17, 18, 21, 22) contain a free radical at the
conserved glycine residue; this radical is generated enzymatically by a
PFL activase and an ARNR activase, respectively. A gene located immediately upstream of the benzylsuccinate synthase structural genes
shares strong predicted amino acid sequence similarity to the PFL- and
ARNR-activating enzymes, suggesting that benzylsuccinate synthase may
be activated in a similar fashion (11, 12).1 In activated
PFL and ARNR, the glycyl radical is assumed to abstract a hydrogen atom
from a conserved cysteine residue generating a thiyl radical that
initiates substrate transformation (14, 15, 20, 23). Genetic
complementation studies in Thauera aromatica sp. T1 showed
that the conserved glycine and cysteine residues of benzylsuccinate
synthase are important for toluene utilization (12). Based on the
molecular similarity of benzylsuccinate synthase to PFL and ARNR, we
examined active benzylsuccinate synthase for the presence of an organic
free radical.
Using EPR spectroscopy we report here the first experimental evidence
that active benzylsuccinate synthase carries a stable organic free
radical. Based on the strong spectral similarities between the EPR
signal of the benzylsuccinate synthase radical and that of the glycyl
radicals of PFL and ARNR, the free radical of benzylsuccinate synthase
appears to be located on a glycine residue. Furthermore we found a
direct correlation between the appearance of the free radical and
enzyme activity, suggesting that the radical is essential for catalysis.
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EXPERIMENTAL PROCEDURES |
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Materials--
All the chemicals used in this study, including
2H2O (D2O; 99.9 atom %) and
[ring-U-14C]toluene (98 atom %; 2.8 mCi/mmol specific activity), were purchased from either Aldrich or
Sigma. The [ring-U-14C]toluene was diluted to
a specific activity of ~42 µCi/mmol for the experiments.
Cultivation of Azoarcus sp. Strain T and Preparation of Cell
Extracts--
Azoarcus sp. strain T, a denitrifying
bacterium capable of anaerobically mineralizing toluene and
m-xylene (24), was cultivated under denitrifying conditions
in a bicarbonate-buffered mineral salts medium as described previously
(1). Batch cultures totaling 10-12 liters were grown in glass reactors
sealed with polytetrafluoroethylene Mininert valves (Alltech
Associates, Inc., Deerfield, IL). These cultures were incubated at room
temperature (25 °C) in an anaerobic glove box (Coy Laboratory
Products, Inc., Grass Lake, MI) with an atmosphere of 80%
N2, 10% H2, and 10% CO2.
Azoarcus sp. strain T was grown exponentially with benzoate
(initial concentration 3 mM) and nitrate (initial
concentration 2.5 mM) to an
A600 of 0.25-0.5. Benzoate and nitrate
were amended repeatedly to the cultures prior to reaching the desired
A600. Approximately 20 h prior to
harvesting the cells, toluene (initial concentration 350 µM) was added to the cultures to induce synthesis of
benzylsuccinate synthase. During the remaining growth period residual
benzoate was consumed completely, and the cells switched to toluene
metabolism as indicated by the induction of benzylsuccinate synthase
activity (data not shown). Cells were harvested anaerobically by
centrifugation (1), washed once in anoxic 120 mM potassium
phosphate buffer, pH 6.8, amended with 2 mM dithiothreitol
(designated as buffer A), and resuspended in 7-12 ml of buffer A
(amended with 12 mg of DNase I and ~6 mM
MgCl2). Cells were broken anaerobically by four passages
through a French pressure cell at 138 megapascal. Unbroken cells
and cell debris were removed by anaerobic centrifugation (27,000 × g, 15 min, 4 °C). The supernatant, defined as the cell extract, was divided into 0.5- and 1-ml fractions that were frozen under anaerobic conditions at 20 °C until further use.
Partial Purification of Benzylsuccinate
Synthase--
Benzylsuccinate synthase was partially purified from
cell extracts of anaerobically grown toluene-induced
Azoarcus sp. strain T using a modification of the method
described previously (10). Purification steps were conducted
anaerobically at ~15 °C in an anaerobic glove box with an
atmosphere of 90% N2 and 10% H2. Crude cell
extract was passed through a 0.45-µm filter and then applied in
batches (35-80 mg of protein) to an Econo-Pac CHT-II hydroxyapatite column (5-ml bed volume, Bio-Rad) attached to an Amersham Pharmacia Biotech FPLC System. The binary eluents used to enrich for
benzylsuccinate synthase activity consisted of a 10 mM
potassium phosphate buffer, pH 6.8, and a 500 mM potassium
phosphate buffer, pH 6.6. Both degassed eluents were amended with 5 mM dithiothreitol. The chromatographic conditions, as
described previously (10), consisted of a 1 ml min1 flow
rate starting at an initial phosphate concentration of 120 mM for 1.2 column volumes and then a linear gradient from
120 to 400 mM phosphate at a rate of 25 mM
min
1. One-ml fractions were collected and assayed for
benzylsuccinate synthase activity as described below. Results from the
hydroxyapatite chromatography were similar to those described earlier
(10) with the following exception: the specific activity of the cell extract was 25 nmol min
1 (mg of protein)
1
and that of enriched benzylsuccinate synthase ranged from 150 to 400 nmol min
1 (mg of protein)
1, depending on
the particular fraction. Benzylsuccinate synthase activity typically
eluted between 160 and 235 mM phosphate. Attempts to purify
benzylsuccinate synthase further by gel filtration resulted in a
greater than 95% loss of specific activity relative to the hydroxyapatite fraction (10). Thus, to retain activity of
benzylsuccinate synthase, hydroxyapatite-enriched fractions were used
for this study. The hydroxyapatite fractions used in this study
contained a greater than 7-fold enrichment of benzylsuccinate synthase
and less than 3% of the protein of the cell extract placed on the column. Protein concentrations were determined by the method of Bradford (25) using a commercially available protein-binding dye from
Bio-Rad. Bovine serum albumin was used as the standard.
In Vitro Assay for Benzylsuccinate Synthase-- The activity of benzylsuccinate synthase was measured using a modified version of the radiological assay developed previously (10). Assays were performed in 2.8-ml glass vials sealed with Mininert valves. The anoxic assay mixtures (final volume 1 ml) contained 100 mM Tris-HCl buffer, pH 7.8, 300 nmol of [ring-U-14C]toluene (specific activity, ~42 µCi/mmol), 10 mM fumarate, and 5 mM dithiothreitol as a reductant. The assay buffer was changed to a Tris-HCl buffer, pH 7.8, because the pH optimum of benzylsuccinate synthase activity was found to be between 7.5 and 8, consistent with the findings of Leuthner et al. (11).
Assays were conducted at room temperature (25 °C) in an anaerobic glove box with an atmosphere of 80% N2, 10% H2, and 10% CO2. To prevent a decrease in the pH of the reaction mixture due to the presence of CO2 in the glove box atmosphere, the reaction mixtures were prepared in a glove box with an atmosphere of 90% N2 and 10% H2 and then transferred to the former glove box in a stoppered anoxic glass bottle. Assay components were added by syringe through Mininert valves. Reactions were started by the addition of crude cell extract or hydroxyapatite-enriched benzylsuccinate synthase. After incubation for 0, 10, or 12 min on an orbital shaker, the reactions were stopped by the addition of 0.1 ml of 2 M NaOH. The assay vials were removed from the glove box and amended with an antifoaming agent (Antifoam A, Sigma; ~100 ppm final concentration). Assay mixtures were purged with N2 for 20 min to remove any residual [14C]toluene. Ultima Gold XR scintillation liquid (10 ml; Packard Instrument Co.) then was added to the mixtures, and the remaining nonvolatile radioactivity (primarily [14C]benzylsuccinate) was measured with a Tri-Carb model 2500 TR/AB liquid scintillation analyzer (Packard Instrument Co.). Benzylsuccinate has been shown to be the only significant product formed from toluene under the experimental conditions of these assays (10).
Sample Preparations for EPR Spectroscopy-- Hydroxyapatite fractions of catalytically active benzylsuccinate synthase were analyzed by EPR spectroscopy. Although a portion of the hydroxyapatite-enriched benzylsuccinate synthase was tested for activity to ensure the enzyme was catalytically active (see above), the remaining portion of benzylsuccinate synthase analyzed by EPR spectroscopy was not amended with the substrates, toluene and fumarate, and therefore did not catalyze the benzylsuccinate synthase reaction. In an anaerobic glove box (atmosphere of 90% N2 and 10% H2), samples (300 µl) of benzylsuccinate synthase were transferred to EPR tubes. Anaerobic tubing was placed over the end of each EPR tube and clamped closed. The EPR tubes were removed from the glove box and immediately frozen in liquid nitrogen. The samples were sent in liquid nitrogen in a shipping Dewar (CryoPak Shipper, CP-100, Taylor-Wharton, Theodore, AL) to Amsterdam where they were analyzed by EPR spectroscopy. Three sample sets of benzylsuccinate synthase were prepared and analyzed by EPR spectroscopy: 1) benzylsuccinate synthase, 2) benzylsuccinate synthase exchanged into phosphate-buffered 2H2O, and 3) benzylsuccinate synthase amended with toluene but not fumarate.
Benzylsuccinate Synthase--
A sample (300 µl) of
hydroxyapatite-enriched benzylsuccinate synthase (1.8 mg of protein
ml1) was transferred to an EPR tube and analyzed by EPR spectroscopy.
Deuterium-exchanged Benzylsuccinate Synthase--
A 200 mM potassium phosphate buffer, pH 6.8, was prepared
anoxically in 99.9% D2O (referred to as phosphate-buffered
D2O). A hydroxyapatite fraction of enriched benzylsuccinate
synthase (800 µl of 1.4 mg of protein ml1) was
exchanged into the anoxic phosphate-buffered D2O by
applying the fraction onto a HiTrap desalting column (5-ml bed volume; Amersham Pharmacia Biotech) and then eluting it with 2.6 ml of the
phosphate-buffered D2O. Only the last 1.9 ml of eluted
liquid were collected, and a portion of this (300 µl of 0.5 mg of
protein ml
1) was transferred to an EPR tube and analyzed
by EPR spectroscopy.
Benzylsuccinate Synthase Amended with Toluene--
Portions (300 µl) of the same hydroxyapatite fraction of benzylsuccinate synthase
(1.9 mg of protein ml1) were added to three EPR tubes.
One EPR tube had no amendment, another was amended with the
nonmetabolizable substrate, benzene (180 nmol), and the third EPR tube
was amended with the substrate, toluene (190 nmol). Both benzene and
toluene were amended from a methanolic stock solution (final methanol
concentration less than 0.7% (v/v)). Fumarate, a cosubstrate of the
benzylsuccinate synthase reaction, was not added to any of the three
samples. The three EPR tubes then were analyzed by EPR spectroscopy.
EPR Spectroscopy--
EPR spectra at X-band (9 GHz) were
obtained with a Bruker ECS 106 EPR spectrometer equipped with an Oxford
Instruments ESR 900 helium-flow cryostat with an ITC4 temperature
controller. A field modulation frequency of 100 kHz was used. The
magnetic field was calibrated with an AEG magnetic field meter. The
microwave frequency was measured with an HP 5350B microwave frequency
counter. The microwave power incident to the cavity was measured with
an HP 432 B power meter. Simulations were carried out as described earlier (26). Quantification of EPR signals was carried out by direct
double integration of the experimental spectra (27, 28) or by
comparison with a good fitting simulation (29).
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RESULTS AND DISCUSSION |
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Benzylsuccinate synthase, as partially purified from extracts of
anaerobically grown toluene-induced cells of Azoarcus sp. strain T, was active and catalyzed the addition of toluene to fumarate
to form benzylsuccinate at high specific activities (150 to 400 nmol
min1 (mg of protein)
1). Using EPR
spectroscopy, we analyzed a hydroxyapatite fraction of benzylsuccinate
synthase (1.8 mg of protein ml
1; ~8 µM
holoenzyme) for the presence of a free radical and observed an EPR
signal as shown in Fig. 2A.
The EPR signal was centered at g = 2.0021 and was characterized by
a 2-fold splitting of about 1.5 mT. The g value of the signal is very
close to that of a free radical. Most importantly, the line shape of
the EPR signal closely resembles that of the glycyl radicals of PFL
(15, 16) and ARNR (17, 18) from E. coli. The EPR signals of
all three enzymes are characterized by a g value between 2.002 and
2.004, as well as a resolved 2-fold splitting of about 1.5 mT (15-18).
The simulation in Fig. 2B shows that the hyperfine
interactions and linewidth proposed for PFL by Wagner et
al. (15) results in an EPR spectrum that is indistinguishable from
that of benzylsuccinate synthase (Fig. 2A). The
EPR spectral similarities between benzylsuccinate synthase and the
glycyl radical enzymes, PFL and ARNR, strongly suggest that the radical
observed in benzylsuccinate synthase is located on a glycine
residue.
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When an EPR sample was thawed under a flow of argon for 15 min and refrozen in liquid nitrogen, the EPR signal did not change (data not shown). However, when the sample was thawed in the presence of air and refrozen in liquid nitrogen, no EPR signal was detected. The absence of an EPR signal is consistent with benzylsuccinate synthase activity being extremely oxygen sensitive (10, 11). The correlation between benzylsuccinate synthase activity and the presence of an EPR signal suggests that the radical is essential for catalysis.
The chemical nature of the radical moiety was investigated further by
exchanging hydroxyapatite-enriched benzylsuccinate synthase into
phosphate-buffered D2O (see "Experimental Procedures")
and analyzing the sample by EPR spectroscopy. The resulting EPR
spectrum revealed no resolved hyperfine splitting (Fig. 2C),
as opposed to the spectrum of the enzyme as determined in
H2O (Fig. 2A). This implies that the 2-fold
splitting observed in the EPR spectrum of the normal protonated enzyme
is caused by a hydrogen atom and that this hydrogen is exchangeable
with deuterium atoms originating from the solvent. Notably, this
hydrogen/deuterium exchange occurs in the absence of catalysis because
the catalytically competent enzyme was not amended with any substrates
prior to analysis by EPR spectroscopy. When PFL was exchanged into
D2O and analyzed by EPR spectroscopy, the resulting EPR
spectrum (15, 16) indicated an analogous behavior. A rapidly
exchangeable hydrogen was found in PFL and was assigned to the position of the glycyl radical (15). Analogous with this assignment,
the 2-fold splitting in the EPR spectrum of benzylsuccinate synthase is
also assigned to an exchangeable hydrogen, most likely the
hydrogen
of the glycyl radical. The simulation in Fig. 2D shows that
the hyperfine interactions and linewidth proposed for PFL in
D2O (15) results in an EPR spectrum indistinguishable from
that of benzylsuccinate synthase in D2O (Fig.
2C). Contrary to benzylsuccinate synthase and PFL, no such
rapidly exchangeable hydrogen was observed for ARNR (17).
Mutant studies conducted with PFL from E. coli showed that
the hydrogen exchange of the glycyl radical of PFL is not spontaneous but involves the active site cysteine 419 (14). Based on this finding,
Parast et al. (14) proposed that the hydrogen of the
glycyl radical does not exchange directly with the solvent. Instead,
the hydrogen of the thiol group of cysteine 419 is proposed to exchange
with the solvent, in this case solvent deuterons, forming a deuterated
thiol group. The glycyl radical is then proposed to abstract the
deuterium from the thiol group, generating a transient thiyl radical
and a chirally deuterated glycine residue. To form a glycyl radical
with a deuterium in the
position, as observed in the
EPR spectra of PFL (15, 16), the thiyl radical of cysteine 419 must nonstereospecifically abstract the hydrogen from the chirally
deuterated glycine residue (14). As an analogy to the findings for PFL,
one may postulate that the hydrogen exchange of the glycyl radical of
benzylsuccinate synthase may not be spontaneous but may involve the
conserved cysteine residue. This would suggest that the glycyl radical
and the conserved cysteine residue in benzylsuccinate synthase are in
close proximity to each other in the active site and interact during catalysis.
We also examined whether amending samples of enriched benzylsuccinate synthase with toluene as the sole substrate alters the EPR signal of the benzylsuccinate synthase radical. Control samples included benzylsuccinate synthase alone and benzylsuccinate synthase amended with benzene, a nonmetabolizable surrogate. The intensity and line shapes of the EPR spectra of all three samples were similar to those of the untreated enzyme (data not shown). This suggests that the presence of toluene alone does not quench the radical to any significant degree, or if it does, the quenching is too transient to be observed.
The spectroscopic characteristics of benzylsuccinate synthase reported
here and the molecular properties discussed previously (10-12, 30)
suggest the following reaction mechanism for benzylsuccinate synthase.
At the beginning of the enzymatic reaction, active benzylsuccinate synthase harbors an oxygen-sensitive, stable glycyl free radical (presumably at glycine 828 in Azoarcus sp. strain T
benzylsuccinate synthase). The glycyl radical abstracts a
hydrogen atom from the conserved cysteine residue forming a
transient thiyl radical (presumably at cysteine 492 in
Azoarcus sp. strain T benzylsuccinate synthase). The thiyl
radical then abstracts a hydrogen atom from the toluene methyl group to
form a benzylic radical as an intermediate, which then attacks the
double bond of fumarate forming a benzylsuccinyl radical. The
benzylsuccinyl radical then reabstracts the same hydrogen atom from the
conserved cysteine residue, forming benzylsuccinate and regenerating
the thiyl radical. Based on proposed reaction mechanisms of PFL (14,
20, 31), we presume that the thiyl radical then abstracts a hydrogen
atom from the conserved glycine residue regenerating the glycyl
radical. At this point the glycyl radical enzyme is competent to
undergo another catalytic cycle. Consequently benzylsuccinate synthase
can undergo multiple turnovers without reintroduction of its glycyl
radical by an activating enzyme, as also indicated by in
vitro assays of benzylsuccinate synthase activity conducted in
this study: 150 pmol of enriched benzylsuccinate synthase produced 160 nmol of benzylsuccinate in 12 min.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grants MCB-9733535 and MCB-9723312.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.
¶ Supported by a grant from The Netherlands Organization for Scientific Research (NWO).
** Recipient of a Terman Fellowship. To whom correspondence should be addressed. Tel.: 650-723-3668; Fax: 650-725-3164; E-mail: spormann@ce.stanford.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009453200
1 G. Achong, A. Rodriguez, and A. Spormann, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PFL, pyruvate formate-lyase; ARNR, anaerobic ribonucleotide reductase; mT, millitesla.
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