2-Difluoromethylene-4-methylenepentanoic acid, a paradoxical probe able to mimic the signaling role of 2-oxoglutaric acid in cyanobacteria

Article abstract of DOI:10.1021/ol2009544

2-Difluoromethylene-4-methylenepentanoic acid (DFMPA), a seemingly deviated analog of 2-oxoglutaric acid (2-OG), could surprisingly mimic its signaling function in cyanobacteria. Computer modeling revealed the favorable binding of DFMPA toward the 2-OG receptor, NtcA, via mutual conformational changes, suggesting that structural alteration of 2-OG is tolerated for it to exercise its signaling role. This extremely useful finding could be exploited for the design of affinity probes with which to study new 2-OG receptors in related signaling pathways.

Full text of DOI:10.1021/ol2009544

ORGANIC
LETTERS
2011
Vol. 13, No. 11
2924–2927
2-Difluoromethylene-4-methylenepentanoic
Acid, A Paradoxical Probe Able To Mimic the
Signaling Role of 2-Oxoglutaric Acid
in Cyanobacteria
Xinjun Liu,†,‡ Han Chen,† Erik Laurini,§ Yang Wang,†,‡ Valentina Dal Col,§
Paola Posocco,§ Fabio Ziarelli, Maurizio Fermeglia,§ Cheng-Cai Zhang,^
Sabrina Pricl,§ and Ling Peng*,‡
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,
ꢀ
P.R. China, Departement de Chimie, CINaM CNRS UPR 3118, 13288 Marseille,
France, Molecular Simulation Engineering Laboratory, DI3, University of Trieste,
ꢀ
34127 Trieste, Italy, Spectropole, Faculte de Saint-Jerome, Marseille, France, and
Laboratoire de Chimie Bacterienne, CNRS UPR 9043, 13402 Marseille, France
ꢀ ^
ꢀ
ling.peng@univmed.fr
Received April 14, 2011
ABSTRACT
2-Difluoromethylene-4-methylenepentanoic acid (DFMPA), a seemingly deviated analog of 2-oxoglutaric acid (2-OG), could surprisingly mimic its
signaling function in cyanobacteria. Computer modeling revealed the favorable binding of DFMPA toward the 2-OG receptor, NtcA, via mutual
conformational changes, suggesting that structural alteration of 2-OG is tolerated for it to exercise its signaling role. This extremely useful finding
could be exploited for the design of affinity probes with which to study new 2-OG receptors in related signaling pathways.
2-Oxoglutaric acid (2-OG, Scheme 1), a metabolite of
the highly conserved Krebs cycle, playsimportant roles not
only in metabolism, but also in the signaling of various
organisms from bacteria1 to plants2 and animals.3 2-OG
forms a carbon skeleton for the assimilation of ammonia
and serves as a lever managing the balance between nitro-
gen and carbon metabolism.4 Using a biological model of
cyanobacteria Anabaena PCC 7120 (hereafter referred to
as Anabaena) and a chemical probe of 2-OG, DFPA
(difluoromethylene pentandioc acid, in Scheme 1), we
revealed the role of 2-OG as a nitrogen starvation signal
in vivo.5 With a difluoromethylene group replacing the
carbonyl moiety, DFPA resembles 2-OG in structure and
is stable to metabolization in vivo. Most importantly,
Scheme 1. 2-OG and Its Analogs DFPA, 2-MPA, and DFMPA
(a hybrid of DFPA and 2-MPA)
† Wuhan University.
‡ CINaM CNRS UPR 3118.
§ University of Trieste.
Spectropole, Marseille.
^ LCB, CNRS UPR 9043.
(1) Ninfa, A. J.; Jiang, P. Curr. Opin. Microbiol. 2005, 8, 168–173.
(2) Feria Benoit, A. B.; Guillot, A.; Ambard Bretteville, F.; Vidal, J.;
Hodges, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 502–507.
(3) Hebeert, S. C. Nature 2004, 429, 143–145.
(4) Hodges, M. J. Exp. Bot. 2002, 53, 905–916.
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(5) Laurent, S.; Chen, H.; Bedu, S.; Ziarelli, F.; Peng, L.; Zhang,
C.-C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9907–9912.
r
10.1021/ol2009544
Published on Web 05/05/2011
2011 American Chemical Society

DFPA can mimic 2-OG, triggering the formation of
nitrogen-fixing heterocysts and inducing a series of cellular
responses by readjusting metabolism in Anabaena.5 Using
another nonmetabolizable probe, 2-MPA (2-methylene-
pentanoic acid, Scheme 1), which employs a vinyl group to
mimic the ketone carbonyl function in 2-OG, we were able
to confirm that it is the ketone form and not the ketal form
of 2-OG that plays a signaling role during nitrogen starva-
tion in Anabaena.6 Subsequent structure/activity relation-
ship analysis with different analogs bearing various
functional groups to mimic the carbonyl group in 2-OG
underlined the importance of this group in its signaling
function.6 Only closely resembling structural motifs such
as difluoromethylene and vinyl moieties could replace the
carbonyl group to ensure the signaling function of 2-OG in
Anabaena, with DFPA and 2-MPA being the best
examples.6
Spurred on by curiosity, we then developed a hybrid
probe based on DFPA and 2-MPA, namely, DFMPA (2-
difluoromethylene-4-methylenepentanoic acid, Scheme 1)
for the structure/activity relationship study. DFMPA
contains both difluoromethylene and vinyl groups at the
C2 and C4 positions, respectively. The dual presence of
these two structural motifs would appear to considerably
change the structure of DFMPA compared to 2-OG.
Consequently, it was expected that DFMPA would neither
resemble 2-OG nor mimic its signaling role. However,
surprisingly DFMPA could mimic the signaling function
of 2-OG to induce heterocysts in Anabaena, in a way
similar to both DFPA and 2-MPA, thus suggesting that
structural alteration may be tolerated for 2-OG to exercise
its signaling role. In order to understand this, computer
modeling was carried out to investigate the interaction
between DFMPA and NtcA, the 2-OG receptor which
senses thenitrogen starvation statusvia changes in the level
of 2-OG in Anabaena.5ꢀ8 Interestingly, DFMPA was
favorably accommodated in the binding site of NtcA via
mutual conformational adaptation, further confirming the
ability of DMFPA to mimc 2-OG and execute its signaling
role. Here, we present our data supporting DFMPA’s
ability to mimic 2-OG signaling in Anabaena and provide
our rationale for the structural alteration on the basis of
computer modeling.
Scheme 2. Synthesis of DFMPA
compositionofDFMPA. However, due to crystal packing,
the crystal structures in the solid state may differ from the
corresponding structures in solution.
We next studied the ability of DFMPA to mimic the
2-OG signaling function in Anabaena. This cyanobacter-
ium is an excellent model to investigate the signaling role of
2-OG in nitrogen metabolism, since it produces morpho-
logically distinct heterocysts in response to combined-
nitrogen deprivation.10 These heterocysts are able to fix
nitrogen from air and hence allow Anabaena to survive
even under nitrogen depletion.11 Importantly, since het-
erocysts differ morphologically from vegetative cells, they
can be easily observed with light microscopy. Further-
more, heterocyst differentiation can be repressed when a
combined nitrogen source such as ammonium or nitrate is
present in the growth medium.
However, DFMPA cannot be taken up efficiently by
Anabaena since it is negatively charged at physiological
conditions.12 We therefore used a recombinant strain of
Anabaena expressing a heterologous 2-OG permease KgtP
from E. coli (referred as KGTP) and which can efficiently
take up 2-OG and its analogs.5,6,12,13 The uptake of
DFMPA in KGTP was studied using High Resolution
Magic Angle Spinning 19F and 1H NMR (HRMAS NMR),
an excellent nondestructive method for the in vivo
analysis of the metabolic profiles of whole cells/tissues.14
Surprisingly, DFMPA could be taken up by the KGTP
strain in a similar way to DFPA and 2-MPA, as demon-
strated by the clear signals observed for gem-difluoro-
methylene at ꢀ105 ppm in 19F NMR (Figure 1A) and
1
the corresponding vinyl group at 5.85 ppm in H NMR
respectively (Figure 1B), whereas, in the control experi-
ments, no such NMR signals were produced by the KGTP
strain or the wild type strain in the absence of DFMPA
(data not shown). The effective uptake of DFMPA by the
KGTP strain suggests its recognition by the 2-OG per-
mease and consequential transporting across the cell
membrane, thus implying that its resemblance with 2-OG
may fool the 2-OG permease. Even more surprisingly was
Synthesis of DFMPA was achieved by coupling ethyl
2-(bromomethyl)acrylate withethyl bromodifluoroacetate
in the presence of activated zinc powder and CuCN,9
followed subsequently by alkali hydrolysis (Scheme 2).
The crystal structure ofDFMPA showsa bent backbone
conformation, which differs from the extended structures
adopted by 2-OG, DFPA, and 2-MPA (Figure S1, Sup-
porting Information). This structural deviation can
be easily understood when considering the chemical
ꢀ
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C.-C.; Peng., L. Chem. Biol. 2006, 13, 849–856.
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Chen, Y. X.; Zhang, C.-C.; Zhou, C. Z. Proc. Natl. Acad. Sci. U.S.A.
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(12) Li, J. H.; Laurent, S.; Konde, V.; Bedu, S.; Zhang, C.-C.
Microbiology 2003, 149, 3257–3263.
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Org. Lett., Vol. 13, No. 11, 2011
2925

Figure 1. HRMAS NMR study on the uptake of DFMPA in the
KGTP strain. (A) 19F and (B) 1H NMR spectra recorded in the
absence (control) and presence of DFMPA.
Figure 3. Effects of 2-OG (A) and its analog DFMPA (B) on the
DNA binding activity of NtcA. Control: DNA fragment with-
out NtcA.
the fact that DFMPA could also induce heterocyst forma-
tion (Figure 2) in the presence of NH4þ, in a manner
similar to DFPA and 2-MPA.5,6 Collectively, these results
suggest that DFMPA might mimic the signaling role of
2-OG in Anabaena.
important for us to understand the tolerance of structural
alteration in 2-OG relating to its signaling role as it could
offer us the opportunity to introduce functional groups in
the design of affinity probes of 2-OG, which are essential in
the identification of other new receptors with a view to
constructingthesignaling pathwaysof 2-OG. Wetherefore
performed computer modeling to obtain detailed informa-
tion on the putative binding modes of DFMPA to NtcA,
with the aim of understanding the structural basis of the
observed biological results.
The modeling procedure, including model building,
protein docking, and free energy calculations, is based on
the docking/Molecular Mechanics (MM)/PoissonꢀBoltzmann
Surface Area (PBSA) approach16 and is described in detail in the
Supporting Information. Briefly, the procedure was developed
and validated by comparing blank tests with the available
X-ray structures of the 2-OG/NtcA and DFPA/NtcA
complexes7 (Figure S3, Supporting Information) and then
applying these to DFMPA and 2-MPA, for which the
crystal structures of their NtcA complexes are not avail-
able. Table 1 displays the estimated binding free energy
(ΔGbind) of DFMPA to the NtcA binding pocket, com-
pared tothecorresponding valuesfor 2-OGand its analogs
Figure 2. Induction of heterocyst differentiation under repres-
sive conditions in the KGTP strain incubated in the absence (A)
and presence (B) of DFMPA. Filaments were incubated with
alcian blue, which specifically stains heterocyst envelope oly-
saccharides (heterocysts are indicated by arrows).
To further confirm that DFMPA mimics the signaling
role of 2-OG in Anabaena, we studied the effects of
DFMPA on the DNA binding activity of the 2-OG
receptor, NtcA,5ꢀ7 a transcription factor which regulates
nitrogen metabolism in cyanobacteria. NtcA is also essen-
tial for the initiation of heterocyst differentiation,8 and
2-OG enhances the DNA binding affinity of NtcA (Figure
3A).15 Similar to 2-OG, DFMPA promoted the DNA
binding of NtcA with increasing concentrations
(Figure 3B) thus implying its ability to mimic the signaling
role of 2-OG via its binding to NtcA in Anabaena.
Furthermore, no heterocyst differentiation was observed
when DFMPA was incubated in the presence of ammo-
nium with the -ntcA KGTP strain, a mutant in which no
NtcA is expressed15 (Figure S2, Supporting Information).
Altogether, these data demonstrate that the heterocyst
formation triggered by DFMPA in Anabaena requires
the 2-OG receptor NtcA, in a way similar to 2-OG.
Table 1. Enthalpy (ΔH), Entropy (ꢀTΔS), and Total Binding
Free Energy (ΔGbind) for 2-OG, DFPA, 2-MPA, and DFMPA
in Complex with NtcAa
2-OG
DFPA
2-MPA
DFMPA
ΔH
ꢀTΔS
ΔGbind
ꢀ50.7 ( 0.4 ꢀ49.6 ( 0.5 ꢀ48.3 ( 0.5 ꢀ46.9 ( 0.4
35.2 ( 0.8
36.0 ( 0.9
35.0 ( 0.8
36.5 ( 0.7
-15.5 ( 0.9
-13.4 ( 1.0
-13.4 ( 1.0
-11.4 ( 0.8
a All values are in kJ/mol. Errors are given as standard errors of the
mean.
DFPA and 2-MPA. From this table we can see that not
only is the applied computational strategy able to yield
fairly accurate absolute free energies of the binding, as
expected, but also, and perhaps more importantly, it can
reproduce the trend exhibited by the corresponding biolo-
gical activity determined for these compounds.
In light of these results, we wanted to know why
DFMPA could maintain the signaling role of 2-OG despite
its deviated structure and how DFMPA could bind toward
the 2-OG receptor NtcA compared with 2-OG. It is
(15) Frias, J. E.; Flores, E.; Herrero, A. Mol. Microbiol. 1994, 14,
823–832.
(16) Pierotti, M. A.; Tamborini, E.; Negri, T.; Pricl, S.; Pilotti, S. Nat.
Rev. Clin. Oncol. 2011, 8, 161–170.
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Org. Lett., Vol. 13, No. 11, 2011

for mutually adapted and thus favorable interactions.
DFMPA changed its conformation when complexed with
NtcA (Figure S4D), similar to that observed for 2-OG on
binding to NtcA7 (Figure S4A). Considerable conforma-
tionalchanges werealsoobservedfor the NtcAbindingsite
(Figures 4): Phe75 displayed a notable rotation of its
phenyl ring, Arg129 underwent a significant translational
move, and the main chain of Leu78, Val77, and Gly76
showed slight rearrangement. All these conformational
changes allowed the favorable and comfortable binding
of DFMPA in the NtcA binding pocket. Mutual adapta-
tion or induced fit17 is a common phenomenon observed in
such as protein/DNA, protein/RNA, protein/protein, and
protein/ligand complexes during their biologicalfunctions.
Here, we have demonstrated that DFMPA also employs
such a mechanism allowing its interaction with NtcA and
assuring the signaling role of 2-OG. This finding is im-
portant as it implies that structural alteration at the C4
position can be tolerated in 2-OG for its signaling function
via interaction with NtcA.
In summary, we have developed the hybrid probe
DFMPA based on our previously developed 2-OG ana-
logs, DFPA and 2-MPA. Our experimental results coupled
with computer modeling have demonstrated that although
DFMPA harbors both difluoromethylene and vinyl
groups at the C2 and C4 positions simultaneously and
has a seemingly important structural deviation compared
to 2-OG, the signaling function of 2-OG is maintained,
inducing heterocysts in Anabaena via the interaction with
the 2-OG receptor, NtcA. Structural deviation of 2-OG is
thus tolerated with respect to its signaling function. How
strict alteration at C4 is allowed for mimicing the signaling
role of 2-OG requires further investigation, for which we
are actively working in this direction. The finding disclosed
here that the tolerance of structural modification at the C4
position in 2-OG is extremely important and may offer us
the opportunity to introduce functional groups in 2-OG
with a view to designing affinity probes of 2-OG. They are
essential means to identifying and studying other 2-OG
receptors with the aim of constructing the signaling path-
ways of 2-OG and increasing our understanding of the
signaling role of 2-OG.
Figure 4. Binding of 2-OG (A) and DFMPA (B) in the binding
pocket of NtcA. H-bonds and salt bridges are highlighted by
black lines. The compounds are shown in atom-colored ball-
and-sticks (C: gray; O: red; F: green). The two H atoms on the
vinyl group are shown as white balls for visualization purposes.
The protein residues directly involved in these interactions are as
follows: Arg88 and Arg129 (blue), Phe75 (light blue), Gly76
(yellow), Val77 (light green), and Leu78 (dark green). All
hydrogen atoms, water molecules, and counterions have been
omitted for clarity.
Figure 4 shows two equilibrated molecular dynamics
structures for 2-OG and DFMPA in complex with NctA.
As we can see, the difluoromethylene group in DFMPA
is located at the space occupied by the carbonyl group of
2-OG, whereas the vinyl group in DFMPA is comfor-
tably accommodated within the NtcA binding pocket.
A close analysis reveals that, for 2-OG (Figure 4A), the
major contribution to NtcA binding stems from two salt
bridges involving the side chains of Arg88 and Arg129
and a network of H-bonds among the main chains of
residues Gly76, Val77, and Leu78. This efficient inter-
molecular interaction scheme is reflected in the affinity
of 2-OG toward NtcA, as testified by the favorable value
of the free energy of binding ΔGbind = ꢀ15.5 kj/mol,
estimated using the MM/PBSA approach. In the case of
DFMPA, the two salt bridge interactions with Arg129
and Arg88, crucial for the binding of 2-OG analogs to
NtcA, have been maintained (Figure 4B), whereas the
MD simulation gives no evidence for the interaction of
DFMPA with Val77 or Leu78. Two stable H-bonds are
however still detected between DFMPA and Gly76.
Thus, the binding mode of DFMPA within the binding
pocket of NtcA is quite similar to that adopted by 2-OG,
although the number and strength of the intermolecular
interactions between DFMPA and its target protein
NtcA are slightly less, in agreement with the correspond-
ing calculated free energy of binding (Table 1). These
molecular modeling studies, coupled with the calculated
affinity of 2-OG analogs toward NtcA, unequivocally
support the apparently surprising experimental finding
that DFMPA can mimic 2-OG via binding to the 2-OG
receptor, NtcA, and thus exercise its signaling role in
Anabaena.
Acknowledgment. We are grateful for the financial
support from Wuhan University, CNRS, and the ANR
PCV program. L.X.J. and W.Y. were supported by the
China Scholarship Council.
Supporting Information Available. Synthesis, NMR
analysis, biological study, and computer modeling as well
as Figures S1ꢀS5. This material is available free of charge
via the Internet at http://pubs.acs.org.
It is important to mention that both DFMPA and the
binding site of NtcA underwent conformational changes
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Int. Ed. 2002, 41, 4706–4708.
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