Mechanism of benzylsuccinate synthase probed by substrate and isotope exchange

Article abstract of DOI:10.1021/ja067329q

Benzylsuccinate synthase catalyzes the first step in the anaerobic metabolism of toluene through an unusual reaction in which toluene is added to fumarate to produce (R)-benzylsuccinate. We have exploited the broad substrate range of the enzyme to demonst

Full text of DOI:10.1021/ja067329q

Published on Web 11/23/2006
Mechanism of Benzylsuccinate Synthase Probed by Substrate and Isotope
Exchange
Lei Li and E. Neil G. Marsh*
Departments of Chemistry and Biological Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1055
Received October 12, 2006; E-mail: nmarsh@umich.edu
Aromatic compounds such as toluene are an important class of
long-lived pollutants that have relatively high water solubility. They
are a particular problem in environments where oxygen is scarce,
such as groundwater supplies, because most pathways to degrade
such compounds require molecular oxygen to functionalize other-
wise inert aromatic hydrocarbons. Recently, however, various
denitrifying and sulfate-reducing bacteria such as Thauera aro-
matica and Desulfobacula toluolica have been discovered that can
live on toluene and related aromatic compounds as their sole source
of carbon and energy under anaerobic conditions.1,2 This discovery
has aroused much interest, because of the potential such bacteria
offer for bioremediation in situations where oxygen is scarce and
because of the novel chemistry involved.3,4
Figure 1. Proposed mechanism for the reaction catalyzed by BSS.
The first step in the anaerobic metabolism of toluene is its
reaction with fumarate to produce (R)-benzylsuccinate.5 This
remarkable reaction is catalyzed by benzylsuccinate synthase (BSS),
which is a member of the glycyl radical family of enzymes that
include pyruvate formate lyase and anaerobic ribonucleotide
reductase.6,7 In the presumed mechanism (Figure 1) the radical is
first transferred to an active-site cysteine that then abstracts a
hydrogen atom from the methyl group of toluene to generate a
benzylic radical. The benzyl radical undergoes addition to the double
bond of fumarate to form a benzylsuccinyl radical, and in the last
step the hydrogen is replaced from cysteine and the glycyl radical
is regenerated.
There is good evidence that the BSS-catalyzed reaction involves
radical intermediates, but its extreme oxygen sensitivity and lability8
have hampered efforts to investigate the mechanism of this unusual
enzyme. We have determined the stereochemistry of hydrogen
transfer from toluene to benzylsuccinate and provided evidence for
the intermediacy of a radical at C-3 of benzylsuccinate.9 We have
recently undertaken a kinetic study of the reaction and through
Figure 2. Representative HPLC chromatograms demonstrating the forma-
isotope effect measurements provided evidence that the formation
of the benzylic radical (3) is most likely the rate-determining step
in the forward reaction.10
tion of HBS from BS and p-cresol. Inset: plot of reaction rate derived from
integration of HBS peak areas.
(disproportionation of BS to toluene and fumarate) must have
occurred. Even though the equilibrium constant for the reverse
reaction is extremely unfavorable, Keq≈ 8 × 10-11 M at 4 °C, the
enzyme catalyzes the reverse reaction at rate only 250-fold slower
than the forward reaction. Furthermore, using deuterium-labeled
BS we observe partial exchange of deuterium with the solvent. This
provides the first direct evidence that the migrating hydrogen is
transferred to a labile site on the protein during catalysis, which is
consistent with the participation of the proposed active site cysteine
residue in the mechanism of BSS.
Measuring the Reverse Reaction. It has previously been shown
that BSS can catalyze the addition of various substituted toluene
derivatives, including p-cresol, to fumarate.12 We developed a
quantitative HPLC-based assay to detect the formation of HBS
catalyzed by BSS in cell-free extracts of T. aromatica (Figure 2).
The assay is very similar to that which we recently described for
assaying BSS activity with its normal substrate, toluene.10
To gain further insight into the mechanism of the BSS-catalyzed
reaction, we have now investigated the ability of BSS to catalyze
the reverse reaction, that is, the formation of toluene and fumarate
from benzylsuccinate. The forward reaction, driven by the formation
of a strong C-C σ-bond at the expense of the weaker C-C π-bond,
is calculated to be exothermic by about 13 kcal/mol.11 This is
comparable to the highly exothermic hydrolysis of phospho-
enolpyruvate to phosphate and pyruvate (∆G° ) -14.8 kcal/mol)
that is generally considered irreversible in the biochemical context.
The exothermic nature of this reaction raises the interesting question
of how the kinetics of the BSS-catalyzed reaction are changed in
the reverse direction.
We have exploited the broad substrate range of the enzyme12 to
demonstrate that BSS can catalyze the exchange of p-cresol with
the benzyl portion of benzylsuccinate (BS) to form (4-hydroxy-
benzyl)-succinate (HBS), indicating that the reverse reaction
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J. AM. CHEM. SOC. 2006, 128, 16056-16057
10.1021/ja067329q CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
In initial experiments, HPLC analysis clearly revealed the
presence of small quantities of HBS in cell-free extracts after
∼24 h incubation at 4 °C with p-cresol (16 mM) and BS (10 mM,
racemic material). The identity of HBS was verified by mass
spectroscopy. This result implied that BSS had catalyzed the
disproportionation of BS to toluene and fumarate, which was then
trapped by reaction with p-cresol to produce HBS. A similar result
was obtained when cell-free extracts were incubated with toluene
and HBS (synthesized enzymatically and purified by HPLC); in
this case BS was recovered from the reaction. Exposure of the cell-
free extract to air abolished the exchange activity. This is consistent
with the exchange reactions being catalyzed by BSS, which, like
all glycyl radical enzymes, is very rapidly inactivated by O2.
In subsequent experiments it was found that the rate at which
HBS was formed by the enzyme from BS and p-cresol could be
significantly increased by rapidly stirring the reaction solution under
a layer of hexane. This is because the hexane extracts the toluene
formed from the aqueous phase, thereby preventing it from
competing with p-cresol, which is not very soluble in hexane. This
allowed us to reliably measure the rate at which HBS was formed
from BS and p-cresol. As shown in Figure 2, the formation of HBS
was linear with time and its rate of formation was 9.7 pmol/min/
mg of protein. Control experiments (see Supporting Information)
in which BS was lacking eliminated the possibility that HBS could
have been formed by the reaction of p-cresol with small amounts
of residual fumarate in the cell-free extract.
We next compared the rate of the exchange reaction with the
rate at which the enzyme catalyzes the formation of HBS directly
from saturating concentrations of p-cresol (16 mM) and fumarate
(10 mM). Under these conditions, HBS was formed at a rate of
420 pmol/min/mg of protein. This is over 40-fold faster than the
exchange reaction measured with p-cresol and BS, indicating that
it is the reverse reaction to form fumarate and toluene that is the
rate-limiting step in the exchange reaction. For toluene reacting
under the same assay conditions, the rate of BS formation was
2400 pmol/min/mg of protein, about 6-fold faster than p-cresol.
Therefore the ratio of the rates for the forward and reverse reactions,
with the substrates at saturating concentrations, is ∼250:1. We note
that this does not allow one deduce anything about the equilibrium
constant for the reaction since the Kms for the substrates are not
known.
Figure 3. Detection of d8- and d7-labeled toluene from the BSS-catalyzed
exchange reaction of d8-labeled BS with p-cresol: (A) GC trace; (B) mass
spectrum of toluene peak.
during the reaction there is a certain probability of a deuterium
exchanging with a proton on the protein or from the solvent. This
observation provides the first experimental evidence that the
migrating hydrogen at C-3 of BS is transferred to a solvent
exchangeable residue on BSS before its subsequent transfer to the
methyl group of toluene. This residue is most likely Cys-492, which
along with Gly-828 is conserved in other glycyl radical enzymes
such as pyruvate formate-lyase and anaerobic ribonucleotide
reductase.13
In the forward reaction, which occurs much more rapidly, we
observed no noticeable loss of isotope when the reaction was
conducted with deuterium-labeled toluene.10 This suggests that in
the reverse direction, conversion of 4 to 3 (Figure 1) occurs
relatively slowly, allowing the active site cysteine time to exchange
a proton with the solvent or other labile site on the protein. In
contrast, in the forward direction, once 3 is formed the remaining
steps in the reaction proceed relatively rapidly, so that the exchange
of protons with the solvent does not have time to occur.
Acknowledgment. We thank Dr. Peter Coschigano (Ohio
University) for advice on the culture of T. aromatica and Christel
Fox and Dr. Mou-Chi Cheng for assistance. This work was
supported by NIH Grant GM 59227 to E.N.G.M.
Supporting Information Available: Details of the preparation of
cell-free extracts, enzyme assay, control experiments, HPLC chromato-
graphs, and mass spectra of compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
Evidence for Thiyl Radical Intermediate in BSS. To directly
confirm the reversibility of the BSS reaction, we aimed to isolate
the toluene produced in the reaction by using BS deuterium labeled
in the toluene portion as the substrate. d8-BS was synthesized
enzymatically from d8-toluene and fumarate and isolated by HPLC.
A reaction containing d8-BS and p-cresol was set up under the
conditions described above and overlaid with hexane. After 24 h,
analysis of the hexane layer by GC-MS clearly identified the
presence of d8-toluene (peaks in the MS with m/z ) 100 and
98 amu), which could only have derived from d8-BS (Figure 3).
Indeed, even in the absence of p-cresol, deuterated toluene could
be detected in the hexane layer, although in much lower abundance.
Thus even though the chemical equilibrium strongly favors the
formation of BS, removing toluene from the aqueous solution is
sufficient to detectably shift the equilibrium toward the reverse
reaction.
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