Studying the signaling role of 2-oxoglutaric acid using analogs that mimic the ketone and ketal forms of 2-oxoglutaric acid.
ABSTRACT 2-Oxoglutaric acid (2-OG), a Krebs cycle intermediate, is a signaling molecule in many organisms. To determine which form of 2-OG, the ketone or the ketal form, is responsible for its signaling function, we have synthesized and characterized various 2-OG analogs. Only 2-methylenepentanedioic acid (2-MPA), which resembles closely the ketone form of 2-OG, is able to elicit cell responses in the cyanobacterium Anabaena by inducing nitrogen-fixing cells called heterocysts. None of the analogs mimicking the ketal form of 2-OG are able to induce heterocysts because none of them are able to interact with NtcA, a 2-OG sensor. NtcA interacts with 2-MPA and 2-OG in a similar manner, and it is necessary for heterocyst differentiation induced by 2-MPA. Therefore, it is primarily the ketone form that is responsible for the signaling role of 2-OG in Anabaena.
Article: Heterocyst development in Anabaena.[show abstract] [hide abstract]
ABSTRACT: Many filamentous nitrogen-fixing cyanobacteria protect nitrogenase from oxygen in differentiated cells called heterocysts. Heterocyst development is controlled by the availability of nitrogen compounds in the environment and by intrinsic factors that regulate the frequency and pattern of heterocysts along vegetative cell filaments. Recent progress in understanding heterocyst development in these simple multicellular organisms includes demonstrating the role of 2-oxoglutarate in regulating the activity of the transcription factor NtcA, the identification of additional genes in the regulatory network, such as hetF, and the further characterization of previously identified genes and proteins, including DevR/HepK, hetR, hetN, patS and patB.Current Opinion in Microbiology 01/2004; 6(6):557-63. · 8.23 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Heterocyst differentiation in filamentous cyanobacteria provides an excellent prokaryotic model for studying multicellular behaviour and pattern formation. In Anabaena sp. strain PCC 7120, for example, 5-10% of the cells along each filament are induced, when deprived of combined nitrogen, to differentiate into heterocysts. Heterocysts are specialized in the fixation of N(2) under oxic conditions and are semi-regularly spaced among vegetative cells. This developmental programme leads to spatial separation of oxygen-sensitive nitrogen fixation (by heterocysts) and oxygen-producing photosynthesis (by vegetative cells). The interdependence between these two cell types ensures filament growth under conditions of combined-nitrogen limitation. Multiple signals have recently been identified as necessary for the initiation of heterocyst differentiation, the formation of the heterocyst pattern and pattern maintenance. The Krebs cycle metabolite 2-oxoglutarate (2-OG) serves as a signal of nitrogen deprivation. Accumulation of a non-metabolizable analogue of 2-OG triggers the complex developmental process of heterocyst differentiation. Once heterocyst development has been initiated, interactions among the various components involved in heterocyst differentiation determine the developmental fate of each cell. The free calcium concentration is crucial to heterocyst differentiation. Lateral diffusion of the PatS peptide or a derivative of it from a developing cell may inhibit the differentiation of neighbouring cells. HetR, a protease showing DNA-binding activity, is crucial to heterocyst differentiation and appears to be the central processor of various early signals involved in the developmental process. How the various signalling pathways are integrated and used to control heterocyst differentiation processes is a challenging question that still remains to be elucidated.Molecular Microbiology 02/2006; 59(2):367-75. · 4.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Can the introduction of fluorine atoms affect the bioactivity of natural semiochemicals? Can fluorine contribute in the creation of specific enzyme inhibitors to interrupt or disrupt the insect communication system? The first step for the bioactivity of a molecule is interaction with the biological sensor. Hydrogen and fluorine are almost bioisosteric and the receptor site of the enzyme can still recognize and accept the fluoro analogue of its natural substrate. However, the peculiar electronegativity of the fluorine atom can affect the binding, absorption, and transport of the molecule. The differences in the molecule's electronic properties can lead to differences in the chemical interactions between the receptor and the fluorinated substrate. Fluorine introduction can modify the metabolic stability and pathway of the semiochemicals in many different ways. Fluorinated analogues can show synergism, inhibition, or hyperagonism effects on insect behaviors, that is, the activity of the nonfluorinated parent compounds can be mimicked, lost, or increased. In any case, the fluorinated molecules can interact with the bioreceptors in a new and disrupting way. The semiochemicals are olfactory substances: fluorine can affect their volatility or smell. Production of semiochemicals from exogenous substances, perception at antennal receptors, and processing of biological responses are the main steps of communication among insects. In the production step, the fluorinated molecules can interact with enzymes that catalyze the biosynthesis of the natural pheromones. In the perception step, fluorinated semiochemicals can interact with the olfactory receptor cells; this often leads to totally unpredictable behaviors. Fluorinated molecules have been developed as probes to elucidate the complex chemorecognition processes of insects. Many of these molecules have been tested to find highly effective behavior-modifying chemicals. New analogues have been synthesized to investigate the metabolic pathway of a pheromone molecule and many of them are promising disrupting agents. Despite such titanic research efforts, the results have often been random, rational trends in the induced behaviors have sometimes been impossible to find, and practical applications of the fluorinated semiochemicals are still uncertain.ChemBioChem 06/2004; 5(5):590-613. · 3.74 Impact Factor
Chemistry & Biology 13, 849–856, August 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.chembiol.2006.06.009
Studying the Signaling Role of 2-Oxoglutaric Acid
Using Analogs that Mimic the Ketone
and Ketal Forms of 2-Oxoglutaric Acid
Han Chen,1,6Sophie Laurent,2,6Sylvie Be ´du,2
Fabio Ziarelli,3Hai-li Chen,4Yong Cheng,5
Cheng-Cai Zhang,2,5and Ling Peng1,4,*
1College of Chemistry and Molecular Sciences
2Laboratoire de Chimie Bacte ´rienne
Institut de Biologie Structurale et Microbiologie
Centre National de la Recherche Scientifique
31 Chemin Joseph Aiguier
13402 Marseille cedex 20
Universite ´s Aix-Marseille I et III
Faculte ´ des Sciences et Techniques de St. Je ´ro ˆme
Avenue Escadrille Normandie-Niemen
13397 Marseille cedex 13
4De ´partement de Chimie
CNRS UMR 6114
163 Avenue de Luminy
13288 Marseille cedex 09
5National Key Laboratory of Agricultural Microbiology
Huazhong Agricultural University
2-Oxoglutaric acid (2-OG), a Krebs cycle intermediate,
is a signaling molecule in many organisms. To deter-
mine which form of 2-OG, the ketone or the ketal
form, is responsible for its signaling function, we
have synthesized and characterized various 2-OG
analogs. Only 2-methylenepentanedioic acid (2-MPA),
which resembles closely the ketone form of 2-OG, is
able to elicit cell responses in the cyanobacterium
Anabaena by inducing nitrogen-fixing cells called het-
erocysts. None of the analogs mimicking the ketal
form of 2-OG are able to induce heterocysts because
none of them are able to interact with NtcA, a 2-OG
sensor. NtcA interacts with 2-MPA and 2-OG in a simi-
lar manner, and it is necessary for heterocyst differen-
tiation induced by 2-MPA. Therefore, it is primarily the
2-OG in Anabaena.
The Krebs cycle is a central metabolic pathway con-
served in all living organisms . It provides not only
the reducing power necessary for the production of re-
spiratory energy, but it also supplies key precursors
for the synthesis of important biological molecules
such as amino acids, lipids, DNA, pigments, etc. One
of its intermediates, 2-oxoglutaric acid (2-OG in Fig-
ure 1A), occupies a strategically important position: it
provides the carbon skeleton required for ammonium
(the reduced form of nitrogen) to be assimilated into
amino acids. 2-OG may therefore be the link between
carbon metabolism and nitrogen assimilation, and it
constitutes acheckpoint for properbalancing ofthecar-
bon/nitrogen metabolisms . It has been suggested for
a long time that 2-OG might constitute a metabolic sig-
nal that regulates the coordination of carbon/nitrogen
metabolism in plants and bacteria [2–4]. It has also
been established recently that 2-OG can serve as a
ligand for an orphan G protein-coupled receptor,
GPR99 , linking metabolism to blood pressure. How-
ever, it is difficult to obtain concrete evidence in vivo
to substantiate this hypothesis because 2-OG is rapidly
metabolized into a variety of molecules. Nonmetaboliz-
able analogs of 2-OG can help us to distinguish its reg-
ulatory functions from its metabolic ones and can pro-
vide valuable information about the function of the
metabolite. We have explored such a strategy and dem-
anobacterium Anabaena sp. strain PCC 7120 (which will
be referred to hereafter as Anabaena) using a nonmeta-
bolizable fluorinated analog, 2,2-difluoropentanedioic
acid (DFPA in Figure 1A) . Since 2-OG constitutes
the carbon skeleton for ammonium assimilation in Ana-
baena, 2-OG accumulates in cells when exposed to
a limitation of a combined nitrogen source in the growth
medium . This transient increased level of 2-OG
serves as a signal to elicit the differentiation of hetero-
cysts, which can fix atomospheric N2to provide a nitro-
gen source for cell growth. The accumulation of DFPA
within cells mimics nitrogen starvation and induces het-
erocyst differentiation even when ammonium is present
. To our knowledge, this study provided the first in
vivo evidence that 2-OG serves as a nitrogen status sig-
nal to induce the formation of a nitrogen-fixing hetero-
eral reasons. First, they demonstrate in vivo that the
Krebs cycle goes beyond its role of metabolism and
that the Krebs cycle intermediates are also involved in
signal transduction. Second, since the signaling func-
tion of 2-oxoglutarate has been suggested in a variety
of organisms from bacteria to human [2–6], the 2-OG
analog could be very useful in a variety of studies.
2-OG may exist in equilibrium between the ketone
form and the hydrated ketal form (Figure 1B), with the
ketone form being predominant. The presence of the
carboxyl group adjacent to the carbonyl group in 2-OG
may favor the formation of the hydrated ketal form.
This prompted us to ask which form of 2-OG is respon-
sible for its signaling role: the ketone form or the ketal
form. DFPA, which has been used previously as an ana-
log of 2-OG, may resemble both the ketone form and the
ketal form: on the one hand, its gem-difluoromethylene
moiety geometrically resembles the tetrahedral ketal
functional group, and on the other hand, its gem-di-
fluoromethylene moiety is held as a good bio-isostere
6These authors contributed equally to this work.
for the ketone functional group [7, 8] due to the special
properties contributed by the fluorine atoms. Therefore,
DFPA is not suitable to offer information as to which
form of 2-OG is responsible for the signaling function.
In order to answer this question, we have developed
various 2-OG analogs (1–12 in Figure 1C) as molecular
probes to study the signaling role of 2-OG. Vinyl analogs
1–4, in which the carbonyl group present in 2-OG was
replaced by the gem-difluorovinyl, vinyl, gem-dichloro-
vinyl, and gem-dibromovinyl groups, respectively,
were designed to mimic the trigonal ketone form of
2-OG; probes 5–12, in which the ketal group present in
the hydrate form of 2-OG was replaced by monohalo-
genmethylene, gem-dihalogenmethylene, as well as cy-
clopropyl, gem-difluorocyclopropyl, and epoxy groups,
were intended to mimic the tetrahedral ketal form of
2-OG. With these analogs, we hoped to probe the struc-
ture-function relationship of2-OG as part ofour ongoing
project focusing on the 2-OG signaling mechanism.
Results and Discussion
Because the unusually strong electronegativity of the
fluorine atom makes the gem-difluorovinyl compound
1 highly reactive and notoriously unstable , we could
not obtain probe 1 in a stable and pure form. We there-
fore gave up our plan to prepare 1. Analogs 2, 5, 6, 8–10,
and 12 were prepared according to the literature reports
[10–14], while 3, 4, 7, and 11 were synthesized using the
methods described in Figure 1D. The synthesis of 3 and
4 was performed via, respectively, dichloromethylena-
tion and dibromomethylenation of dimethyl 2-oxogluta-
rate with triphenylphosphine and the corresponding
tetrahalogenmethane , followed by subsequent hy-
drolysis. Although several methods have been reported
for synthesizing 7 [16, 17], we found them to be ineffi-
cient and inconvenient. We therefore synthesized 7 by
fluorinating diethyl 2-(methylsulfonyloxy)pentanedioate
with CsF , followed by hydrolyzing the correspond-
ing ester to acid. We attemped to use several methods
to synthesize 11. The most straightforward method con-
sisted of performing difluorocarbene insertion into di-
ethyl 2-methylenepentanedioate  and subsequent
hydrolysis. All of the synthesized probes were obtained
in pure forms and gave satisfactory spectral data. They
were soluble in physiological buffers and remained sta-
ble for more than 1 week without degradation.
2-MPA, a Vinyl Analog, Induces Heterocyst
Formation in Anabaena
We then tested the ability of probes 2–12 to mimic the
effect of 2-OG in nitrogen metabolism and cell differen-
tiation using the filamentous cyanobacterium Ana-
baena. Anabaena is a suitable model for investigating
metabolism because Anabaena differentiates a special
type of cells called heterocysts in response to com-
bined-nitrogen deprivation. Heterocysts [20, 21] are
morphologically identifiable cells that can be easily ob-
served under light microscopy. Heterocyst differentia-
tion is repressed when a combined nitrogen source
such as ammonium or nitrate is present in the growth
2-OG levels increased upon deprivation of combined ni-
trogen in Anabaena, and that the nonmetabolizable
2-OG analog DFPA mimics 2-OG and induces heterocyst
formation even under repressive conditions, namely, in
the presence of ammonium [6, 22].
Among all of the synthesized analogs, only the vinyl
analog 2 (2-methylene-pentanedioic acid, 2-MPA) in-
duced heterocyst differentiation in Anabaena (Figure 2)
5–12, which mimic the ketal from of 2-OG, could elicit
acell response in Anabaena. Since 2-MPA is structurally
very similar to the ketone form of 2-OG, but is different
imental finding suggests that it is the ketone form rather
than the ketal form of 2-OG that plays the signaling role
duce heterocyst formation in Anabaena is because they
do not resemble 2-OG in their structure and size due to
the bulky vinyl groups (Figure 3); therefore, they are
unable to be recognized by the 2-OG sensor NtcA in
Anabaena, as described below.
Heterocysts induced by 2-MPA in the presence of
5 mM ammonium were nonfunctional in nitrogen
fixation, since NifH, one of the subunits of the nitroge-
nase complex, was not present, as determined by
Figure 1. 2-Oxoglutaric Acid and Its Various Analogs
(A) 2-Oxoglutaric acid (2-OG) and its analog, 2,2-difluoropentane-
dioic acid (DFPA).
(B) 2-OG may involve an equilibrium between the ketone and the
(C) Proposed analogs of 2-OG to mimic either the ketone form of
2-OG (probes 1–4) or the hydrated ketal form of 2-OG (probes 5–12).
(D) Synthesis of 3, 4, 7, and 11.
Chemistry & Biology
immunodetection withproteins extractedfromcells48hr
after the addition of 2-MPA (data not shown). This sit-
uation is comparable to those reported with heterocysts
formed under similar repressive conditions either in the
mutants[23,24]. Thewholeprocessofheterocyst differ-
entiation, triggered bythe accumulation ofthe2-OG sig-
nal, takes about 24 hr, and the synthesis of the nitroge-
nase only startsaround18 hrafterthe induction.Itis
known that ammonium can switch off nif gene expres-
sion and nitrogenase activity  because it constitutes
the preferred nitrogen source for cyanobacteria, whereas
molecular nitrogen fixation requires high amounts of
ATP and reducing power .
2-MPA Resembles 2-OG in Structure
and in Biological Recognition
To check whether the vinyl analog 2-MPA is able to
mimic the ketone form of 2-OG, we first compared their
molecular structures by analyzing the crystal structures.
Among the vinyl analogs, 2-MPA has a structure very
similar to that of the ketone form of 2-OG (Figure 3):
the length of the C=C bond (1.33 A˚) in 2-MPA is similar
to that of the corresponding C=O bond (1.20 A˚) in
2-OG, and thetwo terminal C–Hbonds ofthe vinyl group
in 2-MPA are equivalent to the two stereochemically im-
the carbonyl group of 2-OG. Replacing the H atom in the
vinyl group present in 2-MPA by Cl and Br significantly
increased the size of the vinyl group in both 3 and 4, re-
sulting in steric hindrance and therefore in structures
and conformations that are different from those of
2-OG (Figure 3).
Inorder toexamine theresemblance andthe bio-isos-
teric effects of 2-MPA in relation to 2-OG, we tested the
inhibitory effect of 2-MPA on L-glutamic dehydrogenase
, an enzyme using 2-OG as a substrate. 2-MPA was
a competitive inhibitor of L-glutamic dehydrogenase
(Figure 4), while 3 showed only weak inhibitory effects
with an IC50value of 1160 mM; no inhibitory effects at
all were observed with 4 and the other analogs (Table 1).
Figure 2. Influence of 2-MPA on Heterocyst Differentiation
(A) The KGTP strain without adding 2-MPA.
(B) Induction of heterocyst differentiation under repressive condi-
tions (in the presence of ammonium) in the KGTP strain incubated
with 2-MPA. Filaments were incubated with alcian blue, which spe-
cifically stains heterocyst envelope polysaccharides (heterocysts
are indicated by arrows).
Figure 3. X-Ray Structures of 2-Oxoglutaric Acid and Its Analogs
(A–E) X-ray structures of (A) 2-OG, (B) 2-MPA, (C) 3, (D) 4, and
2-Oxoglutaric Acid as a Nitrogen Status Signal
These results indicate that 2-MPA, although its vinyl
group is apolar, constitutes a suitable bio-isostere for
the keto function, contrary to the polar but rather bulky
dichlorovinyl and dibromovinyl groups in 3 and 4. More-
over, the similarity between the KMvalue (0.56 mM) ob-
tained with 2-OG and the Kivalue (0.34 mM) obtained
with 2-MPA on the L-glutamic dehydrogenase suggests
that 2-MPA and 2-OG have a similar binding affinity with
this enzyme and confirms that they indeed resemble
each other in many respects.
The structural resemblance between 2-MPA and
2-OG wasfurtherconfirmed bythefactthattheyarerec-
ognized in a similar way by the 2-OG permease KgtP
. Both 2-MPA and 2-OG are negatively charged mol-
ecules at physiological conditions, and they cannot be
taken up efficiently by Anabaena . A recombinant
strain of Anabaena (strain KGTP) expressing KgtP
from E. coli was therefore used . This strain can effi-
ciently take up both 2-OG and DFPA [6, 22]. Like 2-OG
and DFPA, 2-MPA can be taken up by the KGTP strain,
as shown by the results of whole-cell NMR recordings
Spinning NMR) (Figures 5A and 5B), an excellent nonde-
structive method for in vivo studies on the metabolic
profiles and kinetics in whole cells/tissues . 2-MPA
has characteristic1H-NMR signals associated with the
vinyl group at 5.3 and 5.7 ppm. Since the 5.3 ppm signal
was submerged in other NMR signals, we used the
1H-HRMAS NMR (High Resolution Magic Angle
5.7 ppm signal to monitor the uptake of 2-MPA in
KGTP by performing1H-HRMAS NMR (Figures 5A and
5B). Neither the wild-type strain nor the KGTP strain un-
treated with 2-MPA produced this NMR vinyl signal
(data not shown). When the KGTP strain was incubated
with 2-MPA, a clear cut NMR vinyl signal was observed
at 5.7 ppm with increasing incubation time (Figure 5B),
while only a weak signal was recorded in the wild-type
results confirm that 2-MPA enters Anabaena via the
Figure 4. Lineweaver-Burk Plot of the Inhibitory Effects of 2-MPA
on the Activity of L-Glutamic Dehydrogenase
The concentrations of 2-MPA were: 0 M (filled square), 2.0 3 1025M
(filled triangle), and 8.0 3 1025M (filled circle).
Table 1. The Effect of IC50Values of the 2-OG Analogs on the
Activity of the L-Glutamic Dehydrogenase and the pKa Values
of the 2-OG Analogs in Comparison with Those of 2-OG
— 2.35 (2.47)a
aThe data in brackets are reported values for 2-OG.
Figure 5. HRMAS NMR Study on the Uptake of 2-MPA and 7 in the
(A–D) (A and B)1H-HRMAS NMR study on the uptake of 2-MPA in
the KGTP strain and (C and D)19F-HRMAS NMR study on the up-
take of 7 in the KGTP strain. (A)1H-NMR spectra recorded with
the KGTP strain and the wild-type strain in the presence of
2-MPA. (B)1H-NMR spectra recorded with the KGTP strain in the
presence of 2-MPA at the incubation time indicated. (C)19F-NMR
spectra recorded with the KGTP strain and the wild-type strain in
the presence of 7. (D)19F-NMR spectra recorded with the KGTP
strain in the presence of 7 at the incubation time indicated.
Chemistry & Biology
2-OG permease, which provides further evidence that
2-MPA resembles 2-OG.
2-MPA Mimics the Signaling Role of 2-OG
As shown above, 2-MPA can be efficiently taken up
through the 2-OG permease and can induce heterocyst
formation in Anabaena. Furthermore, the in vivo NMR
data obtained with cells incubated with 2-MPA showed
that this molecule can accumulate in the cells, reaching
the maximum level within 7 hr of incubation (Figure 5B),
which corresponds to the level required to trigger het-
erocyst formation in Anabaena, as previously observed
with 2-OG and DFPA [6, 22]. Moreover, the steady accu-
mulation of 2-MPA in cells further suggests that 2-MPA
though we cannot exclude the possibility of minor deg-
radation of 2-MPA in vivo, which cannot be detected
within the limit of the NMR analytical method used here.
To further confirm that 2-MPA mimics the signaling
role of 2-OG in Anabaena, we studied the effects of 2-
MPA on the DNA binding activity of NtcA, a 2-OG sensor
[26, 30, 31]. NtcA belongs to the CRP protein family of
transcription factors and is a general regulator of nitro-
gen metabolism in cyanobacteria including Anabaena
[26, 32, 33]. NtcA is also necessary for the initiation of
heterocyst differentiation [26, 31, 32]. NtcA recognizes
the consensus sequence GTAN8ATC in promoter re-
gions of its target genes and activates their expression,
depending on nitrogen availability . We previously
established that the presence of 2-OG and DFPA en-
hanced the DNA binding affinity of NtcA . The effects
of 2-MPA on the DNA binding activity of NtcA from Ana-
baena were examined by performing DNA mobility shift
tamine synthetase, which is known to be under the posi-
of NtcA to DNA was enhanced with increasing concen-
trations of 2-MPA, as previously observed with 2-OG
and DFPA . However, none of the other analogs,
3–12, had an effect on the DNA binding activity of
NtcA to DNA, as illustrated by the experiment carried
out with 7 (Figure 6B and data not shown). This provides
a further indication that close resemblance between
2-MPA and 2-OG is required for 2-MPA to bind to the
The formation of heterocysts induced by the vinyl
analog 2-MPA was found to require NtcA in vivo, as in
the case of 2-OG and DFPA. The gene ntcA is involved in
the initiation of heterocyst development [20, 26, 32, 34]. The
plasmid bearing the kgtP gene encoding the 2-OG per-
mease  was transferred by conjugation into a DntcA
mutant. As with the KGTP strain, the mutant thus ob-
tained was then incubated with 2-MPA in the presence
of ammonium, but no heterocyst differentiation was
observed (data not shown). These data show that the
differentiation process triggered by 2-MPA in Anabaena
requires NtcA, which is consistent with the results ob-
Structural Requirement for Mimicking
the Signaling Role of 2-OG
designed and synthesized in order to determine which
form of 2-OG (Figure 1B) is responsible for its signal-
ing role. Among these analogs, only the vinyl analog
2-MPA (2), which resembles closely the ketone form of
2-OG in structure and in biological recoginition, is able
to elicit cell responses in the cyanobacterium Anabaena
by inducing heterocysts. This suggests that it is the
ketoneformrather thanthe ketalform of2-OG thatplays
the signaling role in Anabaena. Due to the bulky dichlor-
ovinyl and dibromovinyl groups at C2, vinyl analogs 3
and 4 do not closely resemble 2-OG in structure, and
they are not able to interact with NtcA or to induce het-
erocyst formation in Anabaena (data not shown).
The tetrahedral analogs, 5–12, having various moie-
ties at C2, are intended to mimic the tetrahedral ketal
form of 2-OG. They are structurally different from the ke-
tone form of 2-OG (as shown by 11 in Figure 3E). Indeed,
they are very poor inhibitors of the L-glutamic dehydro-
genase (Table 1), and they do not affect the binding of
NtcA to DNA (7 in Figure 6B and data not shown). They
are therefore poorly recognized by both the 2-OG-re-
lated enzyme L-glutamic dehydrogenase and the 2-OG
sensor NtcA due to the structural difference between
them and 2-OG.
Furthermore, we have examined whether the tetrahe-
dral analogs could be recognized by the 2-OG perme-
ase, which would allow them to enter into cells. We
therefore studied the uptake of 7 and 11 in the KGTP
strain by19F-HRMAS NMR thanks to their characteristic
19F-NMR signals. For the other analogs, we were not
able to follow their uptake process due to the lack of
the convenient and reliable NMR signals. Both 7 and
11 are tetrahedral analogs of 2-OG. Considering the C2
position in these analogs, 7 is less bulky than DFPA,
while 11 is more bulky than DFPA . Results from19F-
HRMAS NMR analysis showed that there was no detect-
able 11 in either the KGTP strain or the wild-type strain
(data not shown), whereas 7 could be taken up by the
KGTP strain (Figure 5D). A less efficient uptake of 7
could also be observed in the wild-type strain (Fig-
ure 5C). By comparing the19F-NMR spectra (data not
Figure 6. Effects of 2-OG and Its Analogs on the DNA Binding Ac-
tivity of NtcA
(A) Organization of the promoter region for glnA; the putative NtcA
binding site is highlighted with bold characters.
(B) DNA binding activity of NtcA toward the promoter region of glnA
in the presence of various concentrations of 2-OG, 2-MPA, and 7.
Control (Co): DNA fragment without NtcA. DNA/protein complexes
are indicated by arrows.
2-Oxoglutaric Acid as a Nitrogen Status Signal
shown), we seethat theuptake of 7inKGTP is,however,
less efficient than that of DFPA. These results indicated
that 7 could be poorly recognized by the 2-OG perme-
ase, while 11 could not be recognized at all by the
2-OG permease. Together with our previous results ob-
tained with DFPA, we can conclude that 2-OG analogs
that closely resemble 2-OG (such as 2-MPA and DFPA)
can be recognized efficiently by the 2-OG permease,
whereas those that do not resemble 2-OG or poorly re-
semble 2-OG, because of the presence of either a too
big (such as 11) or a too small (such as 7) moiety at the
C2 position, are not recognized or poorly recognized
by the 2-OG permease. The fact that 7 could enter into
Anabaena but not induce the heterocyst can be ex-
plained by the absence of an interaction between 7
and NtcA (Figure 6), and the low level of 7 in cells due
to its inefficient uptake may also be a reason for this
lack of induction.
To know whether the pKa value of the carboxylic
group atC1 is also important for mimicking the signaling
role of 2-OG, we have measured the pKa values of all of
the synthesized analogs. As shown in Table 1, the re-
placement of the carbonyl group at C2 by nonpolar moi-
eties (such as in 2-MPA and 10) increases the pKa value
of the carboxylic group at C1, leading to similar pKa
values for the two carboxylic groups at C1 and C5,
whereas the introduction of polar moieties at C2 keeps
their pKa values in line with that of 2-OG. However,
2-MPA is able to mimic 2-OG even though it has a pKa
value of the carboxylic group at C1 that is very different
from that of 2-OG. Therefore, the pKa value of the car-
boxylic group at C1 may not be very important for
2-OG to exercise its signaling role.
Various analogs of 2-OG were synthesized and char-
acterized in this study in order to identify the form of
2-OG responsible for its signaling role in vivo. Only
the vinyl analog 2-MPA, which mimics the ketone
form of 2-OG and resembles 2-OG in many respects,
cyst differentiation in Anabaena. These results lead us
to conclude that it is primarily the ketone form of 2-OG
that is responsible for the signaling function of 2-OG.
Among the 2-OG analogs described in this work, the
chemical modification at the C2 position leads to sig-
nificant structural and biochemical differences be-
tween 2-MPA and the other analogs: 2-MPA closely re-
sembles 2-OG, while all of the other analogs neither
induce heterocyst differentiation in Anabaena nor re-
semble 2-OG due to the presence of either a too big
(such as 11) or a too small (such as 7) moiety at the
C2 position. Therefore, the structural requirment at
C2 is very important for the analogs to mimic the sig-
naling role of 2-OG.
DFPA, the first analog of 2-OG developed in our lab-
oratories, may mimic both the ketone form and the ke-
tal form of 2-OG due to its Janus-faced features con-
tributed by the fluorine atoms. The fact that the vinyl
analog 2-MPA was able to play a similar signaling
role in Anabaena as the gem-difluoromethylene ana-
log DFPA  suggests that both 2-MPA and DFPA
mimic the ketone form of 2-OG. Therefore, both 2-MPA
and DFPA should provide useful means for further
investigations of the signaling role of 2-OG and the
underlying signaling pathways.
Probes 2-MPA, 5, 6, 8–10, and 12 were prepared according to the lit-
erature reports [10–14].
corded at 300 MHz and 75 MHz, respectively, on a Varian Mer-
564.5 MHz on a Varian Mercury-VX600 spectrometer with CF3COOH
as an external standard.1H-HRMAS NMR spectra were recorded at
400 MHz on a Bruker Avance spectrometer. Chemical shifts are re-
ported in parts per million (ppm) with TMS as an internal reference.
FAB-MS was determined using a ZAB-HF-3F mass spectrometer.
L-Glutamic dehydrogenase was purchased from Sigma.
13C-NMR spectra were re-
19F-NMR spectra were recorded at
Synthesis of 3, 4, 7, and 11
Synthesis of 3
Carbon tetrachloride (0.300 ml, 3.15 mmol) was added in a solution
of dimethyl 2-oxoglutarate (0.182 g, 1.05 mmol) and triphenylphos-
phine (1.65 g, 6.29 mmol) in 10.0 ml anhydrous acetonitrile under
Ar at 0?C. Then, the reaction mixture was allowed to warm to
25?C; the color of the solution changed to deep red. After stirring
for 40 min at 25?C, the reaction mixture was extracted with ethyl
ether(20.0 ml), and then the solvent was removed.The obtained res-
idue was purified by column chromatography (petrol ether/ethyl
ether, 20/1) to give the ester product as an oil (0.200 g, 79.0%). A so-
lution of 1 N LiOH (7.36 ml, 7.36 mmol) at 0?C was added slowly to
a solution of this product (0.148 g, 0.610 mmol) in acetone (4.00
ml). After stirring 30 min at 0?C, the reaction mixture was washed
with ethyl ether to get rid of impurities. Then, the aqueous phase
was acidified to pH 1.0 and extracted with ethyl acetate. The organic
phases were combined, evaporated under reduced pressure, and
dried in vacuo, giving 3 as a pale-yellow solid (0.120 g, 90.6%). Crys-
tals were grown by slowly evaporating a solution of 3 in ethyl ether/
hexane.1H-NMR (300 MHz, CD3OD) d (ppm) 2.78 (t, J = 7.7 Hz, 2H),
2.51 (t, J = 7.9 Hz, 2H);13C-NMR (75 MHz, CD3OD) d (ppm) 174.21,
166.81, 132.99, 125.49, 31.45, 28.05; FAB-MS: 213.0 (M, 100.0),
215.0 (M+2, 63.6), 217.0 (M+4, 9.09).
Synthesis of 4
Carbon tetrabromide (1.57 g, 4.74 mmol) was added in a solution of
triphenylphosphine (2.49 g, 9.48 mmol) in 15 ml anhydrous CH2Cl2at
0?C. The color of the reaction solution changed immediately to yel-
low and finally to orange. After stirring for 30 min at 0?C, a solution of
dimethyl 2-oxoglutarate (0.550 g, 3.16 mmol) in 5.0 ml anhydrous
CH2Cl2was added dropwise, and the reaction solution was stirred
at0?C for150min. The reactionmixturewas diluted with 20mlpetrol
ether, and white precipitation appeared immediately. The precipi-
tate was filtrated and washed with ethyl ether. The filtration was
combined and evaporated under reduced pressure. Purification of
the crude residue by chromatography (petrol ether/ethyl ether, 30/1)
gave the ester product (0.660 g, 63.7%) as an oil. A solution of 1 N
LiOH (24.0 ml, 24.0 mmol) at 0?C was added slowly to a solution of
this product (0.660 g, 2.01 mmol) in 20.0 ml acetone. After stirring
for 30 min at 0?C, the reaction mixture was washed with ethyl ether
to get rid of impurities. Then, the aqueous layer was acidified to pH
1.0 and extracted with ethyl acetate. The organic phases were com-
bined, evaporated under reduced pressure, and dried in vacuo, giv-
ing 4 as a yellow solid (0.440 g, 72.5%). Crystals were grown by
slowly evaporating a solution of 4 in ethyl ether/hexane.1H-NMR
(300 MHz, CD3OD) d (ppm) 2.73 (t, J = 7.7 Hz, 2H), 2.51 (t, J = 7.7
141.61, 94.35, 32.08, 31.93; FAB-MS: 301.0 (M, 46.67), 303.0 (M+2,
100.0), 305.0 (M+4, 51.67).
Synthesis of 7
Anhydrous CsF (0.228 g, 1.5 mmol) was dissolved in 5 ml dry
N-methylformamide at 80?C under Ar. A solution of diethyl 2-(meth-
ylsulfonyloxy)pentanedioat (0.141 g, 0.5 mmol) in 2 ml dry N-methyl-
formamide was added to the mixture slowly at this temperature.
After 15 hr at 80?C, the solution was extracted with 20 ml ethyl ether,
and the obtained residue was purified by column chromatography
13C-NMR (75 MHz, CD3OD) d (ppm) 175.32, 169.16,
Chemistry & Biology
to a solution of this product (0.084g, 0.4 mmol) in THF (4.00 ml). After
stirring for 3 hr at 0?C, the reaction mixture was washed with ethyl
ether to get rid of impurities. Then, the aqueous phase was acidified
to pH 1.0 and extracted with ethyl acetate. The organic phases were
combined, evaporated under reduced pressure, and dried in vacuo,
giving 7 as a pale-yellow solid (0.058 g, 97.0%).1H-NMR (300 MHz,
CD3OD) d (ppm) 4.97 (ddd,2JFH= 45.9 Hz,3JHH= 3.9 Hz,3JHH=
3.9 Hz, 1H), 2.48 (m, 2H), 2.10 (m, 2H);
CD3OD) d (ppm) 194.5 (ddd,2JHF= 50.2 Hz,3JHF= 20.3 Hz,3JHF=
Synthesis of 11
Diethyl 2-methyleneglutarate (0.100 g, 0.740 mmol) was mixed with
perature was raised to 110?C. FSO2CF2COOTMS (TFDA) (0.555 g,
2.22 mmol, 0.8 eq/h) was added dropwise, and the reaction was
then stirred at 110?C for 7 hr. The obtained residue was purified by
column chromatography (petrol ether/ethyl ether, 30/1w10/1) to
give the ester product as a yellowish oil (0.111 g, 60.0%). A solution
of 2.5 N LiOH (3.40 ml, 8.50 mmol) at 0?C was added slowly to a so-
lution of this compound (0.121 g, 0.500 mmol) in THF (5.00 ml). The
reaction was warmed to room temperature and was strirred for
2 hr. The reaction mixture was washed with ethyl ether to get rid
of impurities. Then, the aqueous phase was acidified to pH 1.0 and
extracted with ethyl acetate. The organic phases were combined,
evaporated under reduced pressure, and dried in vacuo, giving 11
as a pale-yellow solid (0.091 g, 94.0%), which can be further purified
by crystalization in ethyl ether/hexane.1H-NMR (300 MHz, CD3OD)
d (ppm) 2.47 (m, 2H), 2.28 (m, 1H), 2.18 (m, 1H), 1.80 (m, 1H), 1.56
(t, 1C,1JCF= 285.6 Hz), 34.3 (t, 1C,2JCF= 10.0 Hz), 31.0, 23.6, 20.2
(t, 1C,2JCF= 9.1 Hz);19F-NMR (564.6 MHz, CD3OD) d (ppm) 2137.2
(ddd,2JFF= 150.0 Hz,3JHF= 6.5 Hz,3JHF= 7.1 Hz), 2137.5 (ddd,
19F-NMR (564.6 MHz,
13C-NMR(150 MHz, CD3OD) d (ppm) 175.2, 169.5, 113.3
X-Ray Structural Analysis
Copies of the data can be obtained free of charge upon application
to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Email:
Determination of pKa
A solution of 2 mM 2-OG analog was titrated with 0.1 N NaOH at
room temperature. The variation of pH values was recorded by
a pH meter (METTLER-TOLEDO Delta 320) across the range of pH
values from 2.3 to 12.0. The pKa values for all of the 2-OG analogs
were determined by the nonlinear data processing method. The
pKa values of 2-OG were also determined by this method and
were compared with the reported values.
Inhibition on L-Glutamic Dehydrogenase
The activity of L-glutamic dehydrogenase was monitored spectro-
photometrically at 340 nm and 25?C using a modified protocol pro-
vided by Sigma. The procedure was as follows: aliquots of L-gluta-
mic dehydrogenase (10 ml) and 2-MPA (10 ml) were added to 980 ml
of the assay cocktail, giving the final assay solution containing 88
mM Tris-HCl, 0.94 mM 2-OG, 53 mM NH4OAc, 0.1 mM NADH, 0.25
mM EDTA, at pH 7.3.
For the determination of IC50, the inhibition of the activity of L-glu-
2 mM. The data were plotted using the Microcal Origin 6.0 program.
For the determination of Ki, the inhibition of the activity of L-gluta-
mic dehydrogenase by 2-MPA was measured at four substrate con-
centrations (0.4 mM, 0.5 mM, 0.6 mM, 1 mM) and three inhibitor con-
centrations (0 mM, 20 mM, 80 mM) using the procedure described
above. The Lineweaver-Burk plots were constructed to obtain the
values of KMand Ki.
Culture of the Cyanobacterium Anabaena PCC 7120
The KGTP strain of Anabaena PCC 7120, expressing the 2-OG per-
mease from E. coli, was constructed and cultured as described .
Cells were grown in BG11 medium in the presence of 5 mM ammo-
nium as the nitrogen source. During the exponential phase of cell
growth, CuCl2was added to a 0.8 mM final concentration to induce
erocysts were observed under an optical microscope 24 hr later.
Immunodetection with antibodies against NifH was carried out as
described in . Total proteins were extracted from cells either ex-
posed to a limitation of combined nitrogen for 24 or 48 hr or treated
with 2-MPA in the presence of 5 mM ammonium for 48 hr.
For HRMAS NMR measurement, cells with an OD = 0.4, grown in
BG11 medium with ammonium, were incubated with 1 mM each 2-
MPA, DFPA, 7, and 11, collected at specific times by centrifugation,
and washed twice in BG11 with ammonium.
All NMR spectra were recorded on a 400 MHz Bruker Avance
spectrometer operating at a1H and a19F resonance frequency of
400.1 MHz and 376.8 MHz, respectively.1H and19F experiments
were performed with a commercial 4 mm HRMAS
head, modified in house to observe19F. About 2 mg cell sample
was added to 50 ml D2O, to provide a deuterium lock, and sealed
into a 4 mm Zirconia rotor. To improve the resolution, samples
were spun at Magic Angle, and the spinning rate was set at 4000
Hz. All experiments were recorded at room temperature.
1H-HRMAS NMR spectra were acquired using a CPMG spin-echo
pulse sequence (90 2 [t 2 180 2 t]n2 Acquisition) as a T2filter to
remove the effects of lipids and large macromolecules on spectral
broadening: n = 150; total spin-spin relaxation delay, 2nt = 75 ms.
A 4.8 ms 90?pulse, with 1024 scans and a recycle time of 3 s were
19F-HRMAS MAS NMR spectra were acquired with a single pulse
experiment (SPE) with a pulse length of 6 ms and a recycle delay of 3
s. To obtain a good signal-to-noise ratio, 4600 scans were accumu-
lated for each sample. Chemical shifts were referenced to an exter-
nal standard of 1 M NaF aqueous solution, which has resonance
at 2120 ppm compared with CFCl3.
DNA Binding Activity of NtcA
A 170 bp DNA fragment corresponding to the glnA promoter region
was amplified by PCR with the primers 50-GGATTTTATGTCAAAGTT
GACCCC-30and 50-CGAAACAAAGTTGATGAC-30. The NtcA protein
was purified, and the binding shift assay was performed as previ-
ously described, using 0.18 mg DNA and 0.14 mg protein in a 20 ml
assay system . The DNA bands were visualized after staining
with ethidium bromide.
This work was supported by the Ministry of Science and Technology
in China (Nos. 2003CB114400, 2001CB1089, 2003AA2Z3506), the
National Natural Science Foundation of China, the Cheung-Kong
Scholar Foundation, Wuhan University, and the Centre National de
la Recherche Scientifique. We are grateful to Mr. Michel Giorgi at
the University of Aix-Marseilles III for performing X-ray structural
analysis and to Yi Xia for preparing Figure 3. We thank Dr. Long
Lu, Dr. Fajun Nan, and Dr. Bernard Badet for helpful discussions
Received: January 19, 2006
Revised: May 18, 2006
Accepted: June 1, 2006
Published: August 25, 2006
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Crystallographic data obtained on the structures of 2-MPA, 3, 4, and
11 have been deposited in the Cambridge Crystallographic Data
Center with deposition nos. CCDC 261571, 261399, 261400, and
Chemistry & Biology