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Journal of Bacteriology, October 1998, p. 5454-5457, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of the Novel Benzylsuccinate Synthase
Reaction for Anaerobic Toluene Activation Based on Structural
Studies of the Product
Harry R.
Beller and
Alfred M.
Spormann*
Environmental Engineering and Science,
Department of Civil and Environmental Engineering, Stanford
University, Stanford, California 94305-4020
Received 11 June 1998/Accepted 13 August 1998
 |
ABSTRACT |
Recent studies of anaerobic toluene catabolism have demonstrated a
novel reaction for anaerobic hydrocarbon activation: the addition
of the methyl carbon of toluene to fumarate to form benzylsuccinate. In
vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of
benzylsuccinate. Based on structural studies of benzylsuccinate formed
during anaerobic, in vitro assays with denitrifying,
toluene-mineralizing strain T, we now report the following
characteristics of the benzylsuccinate synthase reaction: (i) it
is highly stereospecific, resulting in >95% formation of the
(+)-benzylsuccinic acid enantiomer
[(R)-2-benzyl-3-carboxypropionic acid], and (ii)
active benzylsuccinate synthase does not contain an abstracted methyl H
atom from toluene at the beginning or at the end of a catalytic cycle.
| |
TEXT |
A novel enzymatic reaction for
anaerobic toluene activation has been reported recently that involves
the addition of the methyl carbon of toluene to the double bond of
fumarate to form benzylsuccinate (Fig.
1). This reaction is of considerable
biochemical interest not only as a novel means of aromatic hydrocarbon
activation but also as a novel means of enzymatic carbon-carbon bond
formation. The benzylsuccinate synthase reaction, which has thus far
been documented in three anaerobic, toluene-degrading bacteria
(denitrifying strain T [2] and Thauera
aromatica [5] and sulfate-reducing strain PRTOL1
[3]), is clearly distinguished from the only class of
reactions previously known to activate aromatic hydrocarbons, oxygenase
reactions, which require molecular oxygen as a cosubstrate (e.g., see
reference 11).
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FIG. 1.
Proposed reactions involved in anaerobic toluene
oxidation to benzoyl-CoA. This pathway is based on research conducted
with denitrifying strain T (2) and T. aromatica
(5) and sulfate-reducing strain PRTOL1 (3).
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In vitro studies with strain T (2) and T. aromatica (5) suggest that benzylsuccinate formation is
the first step of toluene mineralization to carbon dioxide, based in
part on the observed conversion of benzylsuccinate to benzoyl-coenzyme
A (CoA) (a known intermediate of anaerobic toluene mineralization;
e.g., see references 1, 5, 10, and
14) in the presence of nitrate and a source of CoA.
Although all reactions of an anaerobic toluene mineralization pathway
have not been demonstrated, there exists strong evidence for several
reactions depicted in Fig. 1, namely, the toluene-fumarate addition
reaction (2, 3, 5, 12) and the CoA-dependent conversion of
benzylsuccinate to E-phenylitaconate or its CoA thioester
(2) and subsequently to benzoyl-CoA (2, 5).
Intervening reactions in the pathway outlined in Fig. 1 have been
proposed (2, 5).
The reaction mechanism of benzylsuccinate synthase has not yet been
elucidated. However, studies with two Thauera strains suggest that the reaction may be radical, based largely on a high level of homology between the predicted amino acid sequence of the carboxy-terminal region of benzylsuccinate synthase in these Thauera strains and conserved amino acid residues that have
been shown to be essential for the radical mechanism of pyruvate
formate-lyase in Escherichia coli (8, 12). In
order to develop a mechanistic understanding of the toluene-fumarate
addition reaction, we have concentrated on structural analysis of the
reaction product, benzylsuccinate. In previous studies, we discovered
that the H atom abstracted from the toluene methyl group during
addition to fumarate is retained in the succinyl moiety of
benzylsuccinate (2, 3). In this article, we present evidence
that this transferred H atom and the benzyl moiety of a benzylsuccinate
molecule derive from the same parent toluene molecule and that the
toluene-fumarate addition reaction is highly stereospecific,
forming predominantly, if not exclusively, (+)-benzylsuccinic
acid [or (R)-2-benzyl-3-carboxypropionic acid]. The
data reported in this article were generated from anaerobic assays
conducted with permeabilized cells of toluene-grown, denitrifying strain T (2, 9).
Stereospecificity of benzylsuccinate synthase.
Chiral
high-performance liquid chromatography (HPLC) analyses of
benzylsuccinate formed anaerobically in vitro from toluene and fumarate
indicate that only the (+) enantiomer was produced. For these
analyses, benzylsuccinate was solvent extracted from permeabilized-cell assays (1-ml total volume in 20 mM MOPS
[morpholinopropanesulfonic acid] buffer, pH 7.2 [2])
that were amended with toluene (400 nmol), fumarate (500 nmol),
permeabilized strain T cells (~4 mg of protein), and titanium(III)
chloride as a reductant (0.2 mM). The solvent (diethyl ether) extracts
were exchanged into HPLC eluent and analyzed with a Hewlett-Packard
Series 1050 liquid chromatograph. The mobile phase was a 93:7:0.02
(vol/vol/vol) mixture of hexane, ethanol, and trifluoroacetic
acid, respectively, flowing isocratically at 1 ml/min through a
CHIRALPAK AD column (particle size, 10 µm; 250 mm [length] by 4.6 mm [inner diameter]) (Chiral Technologies, Inc., Exton, Pa.). The
injection loop volume was 100 µl. Benzylsuccinate enantiomers were
detected with a wavelength of 254 nm. Authentic (+)-benzylsuccinic acid
[or (R)-2-benzyl-3-carboxypropionic acid] and
(
)-benzylsuccinic acid [or
(S)-2-benzyl-3-carboxypropionic acid] standards (99%
purity) were purchased from Radian International (Austin, Tex.).
In Fig.
2, an HPLC chromatogram of an
ether extract pooled from six permeabilized-cell assays is shown along
with chromatograms
of the same extract coinjected with either a (+)- or
a (
)-benzylsuccinate
standard (0.8 mM). The chiral HPLC chromatograms
demonstrate that
a compound was present in the sample extract that
coeluted with
a (+)-benzylsuccinate standard and that there was no
detectable
compound in the extract that eluted at the retention time of
a
(
)-benzylsuccinate standard. Since gas chromatography-mass
spectrometry
(GC-MS) analysis confirmed that benzylsuccinate was a
predominant
component of this sample extract (data not shown), the
chiral
HPLC results can be interpreted to indicate that
(+)-benzylsuccinate
was produced from toluene and fumarate. If it were
assumed that
(
)-benzylsuccinate was present at just below its
detection limit,
then (
)-benzylsuccinate would constitute less than
5% of the
total benzylsuccinate present in the extract.
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FIG. 2.
Chiral HPLC analysis of benzylsuccinate formed in vitro
from toluene and fumarate. (A) Sample containing benzylsuccinate
produced from toluene and fumarate by permeabilized strain T cells (see
text for experimental conditions); (B) the sample coinjected with a
(+)-benzylsuccinate standard (0.8 mM); (C) the sample coinjected with a
()-benzylsuccinate standard (0.8 mM). The identity of the compound
eluting at ~21 min is unknown.
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As an alternative approach to investigating the stereospecificity of
benzylsuccinate synthesis from toluene and fumarate,
the ability
of permeabilized strain T cells to metabolize (+)-
and
(
)-benzylsuccinate standards was assessed. Previous
studies
have shown that in vitro oxidation of
benzylsuccinate by toluene-degrading,
denitrifying bacteria (strain T
and
T. aromatica) requires the
presence of a source
of CoA and an electron acceptor (
2,
5),
as indicated in
Fig.
1. Preliminary experiments intended to optimize
benzylsuccinate
oxidation in in vitro assays determined that succinyl-CoA
was a better
CoA source than free CoA. In these preliminary experiments,
assay
mixtures (1-ml total volume in 20 mM MOPS buffer; pH 7.2)
containing
racemic commercial benzylsuccinate (150 nmol), nitrate
(2 mM),
permeabilized cells (3 mg of protein), titanium(III) chloride
(0.2 mM), and either CoA (0.3 mM) or succinyl-CoA (0.3 mM) were
incubated
for 1 h, subjected to alkaline hydrolysis to cleave
CoA thioesters
that may have formed, and then extracted, derivatized
with
diazomethane, and analyzed by GC-MS using methods described
previously
(
2,
4). In these experiments, the yields of observed
benzylsuccinate oxidation products (
E-phenylitaconate and
benzoyl-CoA)
were approximately three times greater in the assays
amended with
succinyl-CoA than in the assays amended with free
CoA (data not
shown). Furthermore, in analogous assays amended
with toluene
(400 nmol) and fumarate (500 nmol) rather than with
benzylsuccinate,
E-phenylitaconate and benzoyl-CoA,
yields were approximately four
to six times greater in the
assays amended with succinyl-CoA than
in the assays amended with
free CoA. On the basis of these results,
assays used to investigate
(+)- and (
)-benzylsuccinate oxidation
were amended with
succinyl-CoA rather than with free CoA.
The results of the assays used to investigate (+)- and
(
)-benzylsuccinate oxidation, expressed as nanomoles of
benzylsuccinate,
E-phenylitaconate, and benzoate (or
benzoyl-CoA) detected after
incubation, are presented in Table
1. The tabulated values are
the
averages of duplicate assays (except for the control without
benzylsuccinate, which was not replicated). After incubation,
assay
mixtures amended with (+)-benzylsuccinate contained no detectable
benzylsuccinate (detection limit, ~0.2 nmol). As evidence that
the (+)-benzylsuccinate was metabolized, most of the
initial mass
of benzylsuccinate was recovered as the oxidation products
E-phenylitaconate
and benzoate (accounting for the observed
analytical recovery
of ~50%; Table
1). In contrast, in assay
mixtures amended with
(
)-benzylsuccinate, the mass of
benzylsuccinate recovered after
incubation (50 nmol) was virtually
identical to the amount recovered
in benzylsuccinate-amended controls
that were not amended with
permeabilized cells (52 nmol; data not shown
in Table
1). Furthermore,
the amounts of
E-phenylitaconate
and benzoate found in assay mixtures
amended with (
)-benzylsuccinate
were very low and were similar
to the amounts found in a control to
which no benzylsuccinate
had been added (Table
1); the source of the
small amounts of
E-phenylitaconate and benzoate observed in
the assays amended
with (
)-benzylsuccinate and in the control without
amended benzylsuccinate
was probably residual benzylsuccinate present
in the toluene-grown,
permeabilized strain T cells.
Fate of the methyl H atom abstracted from toluene.
As
reported previously for strains T and PRTOL1, the H atom
abstracted from the toluene methyl group during addition to fumarate is
retained in the succinyl moiety of benzylsuccinate (2, 3). This was substantiated by comparing the electron impact mass spectra of
deuterium-labeled and unlabeled benzylsuccinate formed in vitro from
fumarate and either labeled or unlabeled toluene (2, 3).
We further investigated whether both the H atom abstracted from toluene
and the benzyl portion of its parent toluene molecule
are retained in
the same benzylsuccinate molecule (i.e., whether
or not the benzyl
[C
6H
5CH
2] moiety of
benzylsuccinate and the
abstracted methyl H atom retained in the
succinyl moiety of benzylsuccinate
derive from the exact same toluene
molecule). To examine this
possibility, an anaerobic, 1-h assay was
conducted with an equimolar
mixture of labeled and unlabeled toluene
(~200 nmol each of toluene-
,
,
-
d3 and
unlabeled toluene), fumarate (500 nmol), permeabilized cells
(~3 mg
of protein), and titanium(III) chloride (0.2 mM). After
incubation and
diethyl ether extraction of the assay mixture,
the extract was
derivatized with diazomethane (to form dimethyl
benzylsuccinate) and
analyzed by GC-MS using methods described
elsewhere (
2,
4). The concept underlying this experiment
is as follows. If the
abstracted methyl H (or D) atom remains
with its parent toluene
molecule, then there should be a bimodal
(1:1) distribution of dimethyl
benzylsuccinates with molecular
weights of 236 (C
13H
16O
4; from unlabeled toluene)
and 239 (C
13D
3H
13O
4;
from toluene-
,
,
-
d3). If instead the
abstracted H (or D) atom
does not remain with its parent toluene
molecule, then there should
be a 50% chance of adding either a D
or an H atom to the succinyl
moiety of a benzylsuccinate molecule
containing a benzyl group
from either toluene or
toluene-
,
,
-
d3 (i.e.,
C
6H
5CH
2- or
C
6H
5CD
2-),
resulting in a
tetramodal (1:1:1:1) distribution of dimethyl benzylsuccinates
with
molecular weights of 236, 237, 238, and 239.
The results of this experiment are presented in Table
2. The distribution of benzylsuccinate
was largely bimodal, with a
predominance of molecular
weights of 236 and 239. The relative
distribution of 237-Da
dimethyl benzylsuccinate in the sample
was similar to that in an
unlabeled dimethyl benzylsuccinate standard
(Table
2). The occurrence
of 237-Da dimethyl benzylsuccinate
is consistent with the natural
isotopic abundance of
13C (
13), which can be
used to predict a relative abundance for
237-Da benzylsuccinate of
14.3% (close to the observed 15 to 16%).
An analogous explanation can
be used for the relative abundance
of 240-Da benzylsuccinate, which is
probably a
13C-labeled version of the 239-Da
benzylsuccinate and cannot be
explained by deuterium labeling alone.
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TABLE 2.
Molecular weight distributions of benzylsuccinate
molecules in an unlabeled benzylsuccinate standard and formed
during an in vitro assay
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Concluding remarks.
This article describes salient
characteristics of the unique benzylsuccinate synthase reaction.
Enzymatic addition of toluene to fumarate to yield benzylsuccinate is a
unique reaction with respect to carbon-carbon bond formation as well as
aromatic hydrocarbon activation. Benzylsuccinate synthase differs from
known enzymes that catalyze the formation of new carbon-carbon bonds in
that it catalyzes the addition of a carbon atom to a carbon-carbon double bond rather than to a carbon-oxygen double bond or to
CO2. Benzylsuccinate synthase also differs from
the well-characterized mono- and dioxygenases, which use
molecular oxygen to oxidatively activate aromatic hydrocarbons by
hydroxylation. In contrast, benzylsuccinate synthase activates toluene
by catalyzing its addition to a carboxylated substrate, fumarate. As a
result of the benzylsuccinate synthase reaction, the methyl carbon of
toluene is transformed to a methylene carbon that is in a beta
position to a carboxyl group.
This study, which relies on structural analysis of benzylsuccinate
formed in vitro, reports two observations that provide
insight into the
mechanism of benzylsuccinate synthase. First,
the benzylsuccinate
synthase reaction is highly stereospecific,
resulting in >95%
formation of the (+)-benzylsuccinate enantiomer
[(
R)-2-benzyl-3-carboxypropionic acid]. This suggests that
toluene
adds to the
re face of a C-2 carbon of fumarate,
which is an
sp2-hybridized center. Second, the
benzyl moiety of benzylsuccinate
and the abstracted methyl H atom
retained in the succinyl moiety
of benzylsuccinate derive from the
exact same toluene molecule
(Table
2). This observation suggests that
active benzylsuccinate
synthase does not contain an abstracted methyl H
atom at the beginning
or at the end of a catalytic cycle. In contrast,
examples of carbon-carbon
bond cleaving enzymes that do contain some
fragment of a substrate
molecule before and after a catalytic cycle
include citrate lyase
from
Klebsiella aerogenes and
Streptococcus diacetilactis (
7,
15,
16) as well
as citramalate lyase from
Clostridium tetanomorphum (
6); both enzymes are acetylated in the unbound state.
The proposed reaction mechanism depicted in Fig.
3 is consistent with findings presented
in this study and with suggestions
of other researchers regarding the
possible radical nature of
the benzylsuccinate synthase reaction
(
8,
12). Initially,
activated benzylsuccinate synthase
containing a free radical could
abstract a hydrogen atom from the
methyl carbon of toluene to
yield a benzyl radical (I). The benzyl
radical could then add
to the double bond of fumarate to yield a
(+)-benzylsuccinyl radical
(II). The abstracted H atom retained by
the enzyme could then
react with the (+)-benzylsuccinyl radical (III)
to yield (+)-benzylsuccinate
and the activated enzyme radical (IV). In
vitro studies with purified
benzylsuccinate synthase will be required
to determine the validity
of the proposed radical mechanism.
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FIG. 3.
Proposed reaction mechanism for benzylsuccinate
synthase. E represents the enzyme. Note that the exact same H atom is
bound to the enzyme in II and III.
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ACKNOWLEDGMENTS |
Funding for this study was provided by the National Science
Foundation (MCB-9723312) and by the Office of Research and Development, U.S. Environmental Protection Agency, under grant R-815738 through the
Western Region Hazardous Substance Research Center. Additional support
was provided through an OTL Research Incentive Fund (Stanford University) and a Terman Fellowship to A.M.S.
We thank John Brauman (Stanford University) for helpful discussions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Environmental
Engineering and Science, Department of Civil and Environmental
Engineering, Stanford University, Stanford, CA 94305-4020. Phone: (650)
723-3668. Fax: (650) 725-3164. E-mail:
spormann{at}ce.stanford.edu.
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Journal of Bacteriology, October 1998, p. 5454-5457, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Krieger, C. J., Roseboom, W., Albracht, S. P. J., Spormann, A. M.
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