Thiamine pyrophosphate stimulates acetone activation by desulfococcus biacutus as monitored by a fluorogenic ATP analogue

Article abstract of DOI:10.1021/cb500152y

Acetone can be degraded by aerobic and anaerobic microorganisms. Studies with the strictly anaerobic sulfate-reducing bacterium Desulfococcus biacutus indicate that acetone degradation by these bacteria starts with an ATP-dependent carbonylation reaction leading to acetoacetaldehyde as the first reaction product. The reaction represents the second example of a carbonylation reaction in the biochemistry of strictly anaerobic bacteria, but the exact mechanism and dependence on cofactors are still unclear. Here, we use a novel fluorogenic ATP analogue to investigate its mechanism. We find that thiamine pyrophosphate is a cofactor of this ATP-dependent reaction. The products of ATP cleavage are AMP and pyrophosphate, providing first insights into the reaction mechanism by indicating that the reaction proceeds without intermediate formation of acetone enol phosphate.

Full text of DOI:10.1021/cb500152y

Letters
pubs.acs.org/acschemicalbiology
Thiamine Pyrophosphate Stimulates Acetone Activation by
Desulfococcus biacutus As Monitored by a Fluorogenic ATP Analogue
Olga B. Gutierrez Acosta,†,§ Norman Hardt,‡,§ Stephan M. Hacker,‡ Tobias Strittmatter,‡
́
Bernhard Schink,*,† and Andreas Marx*,‡
‡
†Department of Biology and Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz,
Universitatsstr. 10, 78457 Konstanz, Germany
̈
S
* Supporting Information
ABSTRACT: Acetone can be degraded by aerobic and
anaerobic microorganisms. Studies with the strictly anaerobic
sulfate-reducing bacterium Desulfococcus biacutus indicate that
acetone degradation by these bacteria starts with an ATP-
dependent carbonylation reaction leading to acetoacetaldehyde
as the first reaction product. The reaction represents the
second example of a carbonylation reaction in the
biochemistry of strictly anaerobic bacteria, but the exact
mechanism and dependence on cofactors are still unclear.
Here, we use a novel fluorogenic ATP analogue to investigate
its mechanism. We find that thiamine pyrophosphate is a cofactor of this ATP-dependent reaction. The products of ATP cleavage
are AMP and pyrophosphate, providing first insights into the reaction mechanism by indicating that the reaction proceeds
without intermediate formation of acetone enol phosphate.
cetone is an important solvent used in chemical industry.1
bond between the β/γ-phosphate, or AMP and pyrophosphate
AIts microbial degradation has been studied with aerobic by cleavage of the α/β-phosphate bond. Furthermore, whether
cofactors contribute to the process could not be assayed up to
now.
and anaerobic bacteria. Nitrate-reducing and aerobic bacteria
activate acetone in an ATP-dependent carboxylation reaction
that consumes at least two ATP equivalents per molecule
acetone and forms acetoacetate as a first reaction product.2−5
Desulfococcus biacutus is a strictly anaerobic bacterium that can
grow with acetone as carbon and electron source and sulfate as
electron acceptor.6 Unlike aerobic and nitrate-reducing bacteria,
D. biacutus activates acetone not by carboxylation but uses
carbon monoxide (CO) as a co-substrate in the activation
reaction (Figure 1A). The product of this carbonylation
reaction is acetoacetaldehyde, which is further transformed to
acetoacetyl-CoA and subsequently to two molecules of acetyl-
CoA.7 The mechanism of the novel carbonylation reaction is
still under investigation.
The product of the carbonylation reaction, acetoacetalde-
hyde, is highly unstable, and therefore could be identified only
as the dinitrophenylhydrazone derivative or after derivatization
with guanidine leading to 2-amino-4-methylpyrimidine.7 Differ-
ent from nitrate-reducing and aerobic bacteria2−5 it is unlikely
that D. biacutus invests two ATP equivalents into this activation
reaction because the overall energy budget of sulfate-reducing
bacteria is very tight. Nevertheless, acetone activation by sulfate
reducers does require some energy input by ATP hydrolysis in
the initial step, as shown recently.7
In this study, using novel fluorogenic ATP analogues, we
found that thiamine pyrophosphate (TPP) plays a crucial role
in the transformation of acetone by D. biacutus. Furthermore,
we found that the products of ATP cleavage are AMP and
pyrophosphate, indicating that acetone enol phosphate, a
conceivable reaction intermediate, is not formed.
For the investigation of acetone transformation in cell-free
extracts (CE) we envisioned to utilize an ATP-based Forster-
̈
resonance energy transfer (FRET)-cassette that should monitor
ATP consumption in real time as recently reported in studies
on isolated enzymes.8,9 In this concept, an ATP analogue is
intramolecularly equipped with a fluorescent donor at the γ-
phosphate and an acceptor attached to the nucleoside part of
ATP (Figure 1B). In the noncleaved state, the excited donor
fluorophore can transfer its energy to the acceptor via FRET. If
a nonfluorescent acceptor dye (dark quencher) is used, this
results in a nonfluorescent ATP analogue. Upon cleavage of the
probe by an enzyme, the acceptor dye is spatially separated
from the donor dye, and the specific fluorescence emission of
the donor is restored.
In order to employ these analogues as activity probes toward
ATP-dependent processes in this specific study on CE of D.
Due to the instability of the product acetoacetaldehyde, the
activating reaction is difficult to characterize. Therefore, it is not
known whether ATP is hydrolyzed during this process to result
in ADP and phosphate by cleavage of the phoshoranhydride
Received: February 27, 2014
Accepted: April 29, 2014
Published: April 29, 2014
© 2014 American Chemical Society
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Figure 1. Investigation of the novel carbonylation reaction of acetone in D. biacutus. (A) Pathway proposed by Gutierrez Acosta et al. involving ATP-
and CO-dependent acetone activation by Desulfococcus biacutus.7 (B) Modified ATP probe, optimized for experiments with cell-free extracts, bearing
a fluorescent donor D attached to the γ-phosphate and a quencher Q attached to the C2-position of the nucleobase.
Figure 2. Synthesis and fluorescence characteristics of probe 1. (A) Compound 2 was synthesized as described.9 Reagents and conditions: a) Eclipse-
quencher-NHS, 0.1 M NaHCO3 (pH 8.7), DMF, rt, overnight, 3 in 26%. b) TCEP, H2O/MeOH/NEt3, rt, 4 h, 4 in 57%. c) SulfoCy3-NHS, 0.1 M
NaHCO3 (pH 8.7), DMF, rt, overnight, 1 in 80%. (B) Reaction of the fluorogenic ATP activity probe 1 with phosphodiesterase I (SVPD) of C.
adamanteus. (C) Fluorescence emission spectra of the ATP probe 1 without SVPD (black) and after digestion with SVPD (gray); excitation
wavelength was 532 nm. (D) RP-HPLC analysis of the negative control without SVPD (left) and the SVPD-digested ATP probe 1 (right) were
performed according to the general procedures. After 30 min of incubation quantitative conversion is detected. Fractions of negative and positive
control were identified by high resolution mass spectrometry (HRMS).
biacutus, it is required to ensure its stability in CE. It was shown
recently10 that phosphoester modifications are stable in a wide
pH range and are therefore suited to investigate enzyme
reactions. However, all recently reported ATP-based FRET-
cassettes8,9 were not stable in CE of D. biacutus. We found that
the fluorogenic ATP analogue 1 was suited for the analysis of
enzymatic reactions performed with CE of D. biacutus. The
novel probe carries a Sulfo-Cy3 dye at the γ-moiety of the
triphosphate and an eclipse-quencher connected to the C2-
position of the nucleobase.
The synthesis of probe 1 starts with the modified adenosine
triphosphate 2 that was reported before9,11 (Figure 2A). The
eclipse-quencher was introduced by treatment of the free amine
with the corresponding N-hydroxysuccinimide (NHS) ester of
the dye to form 3 with 26% yield. The azide in compound 3
was reduced using tris(2-carboxyethyl)phosphine (TCEP),
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Letters
generating the amino group to form 4 in 57% yield. Finally,
Sulfo-Cy3 was introduced as described above using NHS ester
chemistry, resulting in ATP analogue 1 with 80% yield.
To evaluate the optical properties of 1, fluorescence spectra
of the noncleaved and cleaved state were measured. The
cleavage of probe 1 between the α- and the β-phosphate was
promoted using snake venom phosphodiesterase (SVPD) of
Crotalus adamanteus (C. adamanteus) that is known to
efficiently cleave this type of molecule and liberate both dyes
of the FRET cassette8 (Figure 2B−D). The experiment shows
that upon cleavage of the donor dye from the acceptor the
fluorescence intensity changes dramatically, rendering the
probe suitable as a nucleotide-based activity probe toward
ATP-consuming enzymes.
cofactor in acetone activation by D. biacutus. The hydrolysis
rate of probe 1 was two times higher when the protein
concentration was doubled (data not shown). The hydrolyzing
activity of 1 depended furthermore on acetone and CO,
indicating the specificity of the reaction. The ATP analogue 1
was cleaved to a minor extent also by other ATP-cleaving
enzyme(s) present in the CE, as indicated by the background
activity measured in the absence of acetone (Figure 3A).
We next investigated the effect of the subsequent oxidation
of acetoacetaldehyde to acetoacetyl-CoA on the rate of
hydrolysis of probe 1 (Figure 3B). Addition of CoA and
NAD+ to the reaction mix containing TPP caused a further
increase of the activity (28.7
3.11 nmol min−1 mg−1 total
protein), but no significant increase was observed after addition
of only CoA or only NAD+. This rate is comparable with the
rate of the acetone activation reaction in the presence of
nonmodified ATP; a specific activity of 40.1 nmol min−1 mg−1
total protein was measured for acetoacetaldehyde formation
with subsequent reaction with guanidine.7 Furthermore, it is
even higher than the specific substrate turnover rate of D.
biacutus cells growing with acetone and sulfate (19 nmol min−1
mg−1 total protein as calculated from the doubling time and the
substrate-specific growth yield).12 Compared to a TPP-free
control with CoA plus NAD+, TPP addition caused even a 7-
fold stimulation of the overall reaction.
We next set out to use 1 to study acetone carbonylation in
extracts of D. biacutus (Figure 3A and B). We detected a linear
The increase in the hydrolysis rate of probe 1 after addition
of CoA and NAD+ is consistent with the novel pathway for
acetone degradation that we proposed before,7 in which the
formed acetoacetaldehyde is oxidized further to acetoacetyl-
CoA with CoA and NAD+ as cofactors. Oxidation of
acetoacetaldehyde after acetone activation was also shown
before by mass spectrometry.7 In the absence of these cofactors,
the activity is impaired, perhaps due to accumulation of the
highly reactive acetoacetaldehyde. Reactive aldehydes are highly
toxic because of their ability to form adducts with functional
groups of proteins.13,14
To further characterize the fate of probe 1 during the
enzymatic reaction, RP-HPLC analysis of the reaction mixtures
was performed. In the absence of CO only probe 1 could be
detected (Figure 3C). This shows that probe 1 is stable to the
applied conditions and can be reliably applied in experiments
with CE. When the reactions were performed in the presence of
CO a single peak eluted at the retention time and with the
spectral properties of compound 5. The corresponding cleavage
product 6 could not be detected, possibly due to its low
absorbance. The identity of cleavage product 5 was further
proven by HRMS. Production of 5 bearing the diphosphate
moiety shows that ATP is cleaved to AMP and pyrophosphate
in the process of acetone carbonylation and reveals the mode of
ATP cleavage during this process. Cleavage of ATP to AMP
and pyrophosphate indicates that acetone is not phosphory-
lated to phosphoenol-acetone in this reaction. However,
formation of acetonyl-AMP as an intermediate cannot be
ruled out at the moment.
Our findings strongly indicate that TPP assists the acetone
activating enzyme(s) in D. biacutus, by acting as a cofactor in
the initial reaction. Furthermore, we also show that acetone is
not phosphorylated by ATP to give phosphoenol-acetone as
ATP is cleaved to yield AMP and pyrophosphate. Although the
mechanism of TPP involvement in acetone activation is still
unclear, one may speculate that TPP forms an intermediate
with acetone or an acetone analogue that has been previously
activated by ATP. Alternatively, one may speculate a reaction
Figure 3. Reaction of ATP analogue 1 in cell-free extract (CE) of D.
biacutus. All experiments were performed under anoxic conditions at
30 °C. Concentrations of the substrates were 1 mM acetone, 0.5 mM
ATP probe 1, 2 mM TPP, 2 mM CoA, 4 mM NAD+, and 10% CO in
the headspace. (A) Time course experiments of the hydrolysis of ATP
probe 1 in the acetone activation reaction. (B) Activity of hydrolysis of
ATP probe 1 with addition of cofactors after 60 min of enzymatic
reaction. (C) RP-HPLC analysis after 60 min of the control under
nitrogen (top) gave intact ATP probe with a retention time of 21 min;
the positive control (bottom) showed cleavage product 5 with a
retention time of 16 min and typical spectral properties. Fractions of
negative and positive control were further identified by high resolution
mass spectrometry.
increase of Sulfo-Cy3 fluorescence over time in the presence of
CE, acetone, and CO. The rate of 1 consumption was
determined to be 2.1 0.47 nmol min−1 mg−1 total protein.
In order to gain further insight into the reaction mechanism,
we checked for a possible requirement of cofactors (Figure 3A
and B). Initial analysis in our laboratory indicated that TPP
might be involved in the transformation of acetone. Thus, we
investigated a possible involvement of TPP as cofactor in the
metabolism of acetone. TPP addition caused a nearly 4-fold
increase of the rate of hydrolysis of probe 1 (7.77 0.27 nmol
min−1 mg−1 total protein), which indicates that TPP acts as a
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Letters
strains and comparison of acetone carboxylase enzymes. Appl. Environ.
Microb. 77, 6821−6825.
between TPP and CO and a subsequent addition to (activated)
acetone. In any case, the reaction would result in the
consumption of only one ATP equivalent for the synthesis of
acetoacetyl-CoA, whereas acetone-carboxylating aerobic and
nitrate-reducing bacteria have to invest at least two ATP
equivalents to obtain the same product. This would be
favorable for a sulfate-reducing bacterium with its very tight
energy budget. The reaction mechanism is to be further
elucidated in our lab, with special focus on a TPP-
acetoacetaldehyde intermediate.
Conclusions. In the present study, we investigated the
acetone carbonylation reaction in D. biacutus using a novel
fluorogenic ATP analogue optimized for experiments in cell
extracts. We find that ATP is cleaved to AMP and
pyrophosphate, and that thiamine pyrophosphate is a cofactor
that enhances the enzymatic acetone activation. These findings
suggest that phosphorylation of acetone to phosphoenol-
acetone is not involved in this specific enzymatic reaction, but
acetone is probably activated by ATP in a different manner
resulting in the formation of AMP and pyrophosphate.
(5) Schuhle, K., and Heider, J. (2012) Acetone and butanone
̈
metabolism of the denitrifying bacterium ″Aromatoleum aromaticum″
demonstrates novel biochemical properties of an ATP-dependent
aliphatic ketone carboxylase. J. Bacteriol. 194, 131−141.
(6) Platen, H., Temmes, A., and Schink, B. (1990) Anaerobic
degradation of acetone by Desulfococcus biacutus spec. nov. Arch.
Microbiol. 154, 355−361.
(7) Gutierrez Acosta, O. B., Hardt, N., and Schink, B. (2013)
Carbonylation as a key reaction in anaerobic acetone activation by
Desulfococcus biacutus. Appl. Environ. Microb. 79, 6228−6235.
(8) Hacker, S. M., Pagliarini, D., Tischer, T., Hardt, N., Schneider, D.,
Mex, M., Mayer, T. U., Scheffner, M., and Marx, A. (2013)
Fluorogenic ATP analogues for online monitoring of ATP
consumption: observing ubiquitin activation in real time. Angew.
Chem., Int. Ed. 52, 11916−11919.
(9) Hardt, N., Hacker, S. M., and Marx, A. (2013) Synthesis and
fluorescence characteristics of ATP-based FRET probes. Org. Biomol.
Chem. 11, 8298−8305.
(10) Hacker, S. M., Mex, M., and Marx, A. (2012) Synthesis and
stability of phosphate modified ATP analogues. J. Org. Chem. 77,
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(11) Hacker, S. M., Hardt, N., Buntru, A., Pagliarini, D., Mockel, M.,
Mayer, T. U., Scheffner, M., Hauck, C. R., and Marx, A. (2013)
Fingerprinting differential active site constraints of ATPases. Chem. Sci.
4, 1588−1596.
METHODS
■
Full experimental details are given in the Supporting Information.
ASSOCIATED CONTENT
(12) Janssen, P. H., and Schink, B. (1995) Catabolic and anabolic
enzymes activities and energetics of acetone metabolism of the sulfate-
reducing baterium Desulfococcus biacutus. J. Bacteriol. 177 (2), 277−82.
(13) Schauenstein, E. (1967) Autoxidation of polyunsaturated esters
in water: chemical structure and biological activity of the products. J.
Lipid Res. 8, 417−428.
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
(14) Esterbauer, H., Zollner, H., and Scholz, N. (1975) Reaction of
glutathione with conjugated carbonyls. Z. Naturforsch. C 30, 466−473.
■
Corresponding Authors
*E-mail: Bernhard.Schink@uni-konstanz.de.
*E-mail: Andreas.Marx@uni-konstanz.de.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge funding by the Deutsche For-
schungsgemeinschaft within the SFB 969 and the SPP 1319
priority program, the Studienstiftung des Deutschen Volkes and
the Zukunftskolleg of the University of Konstanz for a stipend
to S.M.H. and the Konstanz Research School Chemical Biology
for fellowships granted to O.B.G.A., N.H., and S.M.H. We
thank A. Wiese for preparation of bacterial growth media.
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