livret final - Institut des Métaux en Biologie de Grenoble

Transcription

9èmes Journées Scientifiques
de l’Institut des Métaux
en Biologie de Grenoble
Autrans - 22 et 23 Mai 2014
2
Le mot du Directeur
Chers amis,
C’est aujourd’hui la 9ème édition des Journées de l’IMBG qui ont rythmé au fil du temps la vie de
l’Institut. Ces journées, au départ annuelles, ont, depuis 2004, été organisées en alternance avec le
congrès international. Elles n’ont pu avoir lieu l’année dernière en raison de l’organisation du congrès
ICBIC XVI, qui s’est tenu à Grenoble en Juillet 2013. Ces réunions sont le ciment de notre activité et il
est important que tout le site grenoblois y soit impliqué. C’est pourquoi il nous faut continuer à
transmettre le message de l’Institut autour de nous et d’ouvrir notre périmètre à d’autres horizons. Par
exemple, des laboratoires de l’Université de Savoie seraient intéressés pour nous rejoindre. Il est aussi
envisageable si nous le souhaitons de se tourner vers les autres universités de la région.
Notre mission reste de favoriser les échanges de chercheurs et d’enseignants-chercheurs dont le
point commun est un projet scientifique : la Chimie et la Biologie des métaux dans les systèmes vivants
et leurs applications dans le domaine de la catalyse, de l’environnement et de la santé. Les thématiques
évoluant très vite, on s’aperçoit que la chimie durable, la biologie de synthèse et la chemobiologie
seront les disciplines de demain. La connaissance, le contrôle et la bio-inspiration des mécanismes liés
aux métaux restent alors incontournables et nous avons donc notre place pour participer à cette
dynamique. La mission de formation de l’institut est parfaitement remplie cette année avec la quasitotalité des communications orales présentées par des doctorants ou post-doctorants.
Nous arrivons aujourd’hui dans une période d’évaluation et la place de l’Institut ne semble pas être
remise en question. Nous conservons la confiance de l’Université qui a souvent présenté l’institut
comme un exemple réussi de structuration scientifique. Néanmoins devant les contraintes budgétaires,
il faudra sans doute faire preuve d’inventivité pour assurer l’équilibre des budgets de nos
manifestations, notamment du 6eme congrès international et de son école thématique dédiée qui se
profilent.
Je vous invite à discuter du futur de l’Institut et du choix des manifestations pour l’année 2015 au cours
de l’assemblée générale.
Je vous remercie pour votre participation et vous souhaite à tous une bonne réunion et des discussions
scientifiques les plus prolifiques.
Pensez à visiter le site : http://imbg.ujf-grenoble.fr. Vous y retrouverez les missions de l’Institut.
Grenoble le 6 Mai 2014
Au nom du comité de pilotage
Stéphane Ménage
Directeur de l’IMBG
3
4
PROGRAMME
5
6
Jeudi 22 Mai
9h00
Accueil
9h15
Ouverture par Stéphane MENAGE
9h20-12h30
Conférences
Modérateur : Nicolas LECONTE
9h20
Conférence invitée : Clotilde POLICAR
Single core multimodal probes for imaging (SCoMPIs): classical fluorescence, IR-mappings and
quantification
10h10
Stéphanie RAT
Superoxide reductase as a model for oxygen activation
10h30
Pause
11h00
Vincent LEBRUN
Efficient oxidation and destabilization of zinc fingers by singlet oxygen
11h20
Florence PUCH
Sodium selenite induced an apoptotic cell death process and exhibited anti-tumorigenic properties in
Glioblastoma cells: Monolayer (2D) and multicellular spheroid cultures (3D) comparisons
11h40
Bertrand GEREY
New homo- and heteronuclear complexes of Manganese/Calcium: spectroscopic models for the
investigation of calcium in the Oxygen-Evolving Center of Photosystem II
12h00
Alice CHAN
NAD biosynthesis in procaryotes: Structural and functional studies of the Quinolinate Synthetase
12h15
Juan FONTECILLA-CAMPS
The crystal structure of FeS quinolinate synthase unravels an enzymatic dehydration mechanism that uses
tyrosine and a hydrolase-type triad
12h30
Déjeuner
7
14h00-16h00
Posters
16h00-19h00
Conférences
Modérateur : Vincent NIVIERE
16h00
Conférence invitée : Wolfgang NITSCHKE
Thermodynamics + redox couples + metals = Life
16h50
Charlène ESMIEU
N2O reduction at a mixed-valent {Cu2S} center
17h10
Pause
17h30
Esther CASTILLO
Visible light hydrogen production in water from a cobalt(III) tetraaza-macrocyclic catalyst
17h50
Widade ZIANI
Transmembrane signal transduction by the CnrYXH complex in Cupriavidus metallidurans CH34:
interaction between CnrX and CnrY
18h10
Conférence invitée : Christelle HUREAU
Cu and Zn impacts in Alzheimer's disease
19h30
Apéritif
20h00
Dîner
21h30-23h00
Assemblée générale
8
Vendredi 23 Mai
8h30-12h10
Conférences
Modérateur : Jacques COVES
8h30
Conférence invitée : Olivier NEYROLLES
Metallobiology of host-pathogen interactions: an intoxicating new insight
9h20
Emeline SAUTRON
Biochemical characterization of AtHMA8/PAA2, a chloroplast-thylakoid Cu(I)-ATPase
9h40
Doti SERRE
Artificial inorganic nucleases involving the phenolate-copper entity
10h00
Wissam IALI
Photocatalytic oxidation of organic substrates by Ru/Cu hetero-metallic complexes with dioxygen as the
unique oxygen atom source
10h20
Pause
10h50
Jordi RULL-BARULL
Grafting of 3,4-epoxybutyltrimethoxysilane on silicon oxide by supercritical carbon dioxide
11h10
Roman ROHAC
Structural insights into a novel radical based carbon-sulfur bond formation in radical SAM enzyme
HydE from Thermotoga maritima
11h30
Nicolas KAEFFER
Water splitting: from bio-inspiration to system integration
11h50
Laurianne RONDOT
A Ru based artificial oxydase
12h10
Déjeuner
14h00-15h50
Conférences
Modérateur : Géraldine SARRET
14h00
Conférence invitée : Olivier MAURY
Original lanthanide luminescent bioprobes for new biphotonic microscopies
14h50
Manon ISAAC
Lanthanide-based luminescent probe for time-gated detection of copper(I) : modulation of the antenna effect
by cation/̟ interaction
15h10
Matteo COLOMBO
Cobalt regulation of the PfTET3 aminopeptidase activity
15h30
Eve de ROSNY
Structural and functional analysis of the Ferric Uptake Regulator (FUR) proteins from various pathogens
15h50
Clôture
9
10
CONFERENCES
&
COMMUNICATIONS
ORALES
11
12
Single Core Multimodal Probes for Imaging (SCoMPIs):
classical fluorescence, IR-mappings and quantification
C. Policar,a,b,c S. Clède,a,b,c F. Lambert,a,b,c H. Bertrand,a,b,c N. Delsuc,a,b,c C. Sandt,e Z. Gueroui,a,b,d
M.-A. Plamont,f A. Vessières,f A. Dazzig
a) Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24 rue Lhomond F-75005 Paris, France
b) Sorbonne Universités, UPMC Univ Paris 06, F-75005 Paris, France
c) CNRS, UMR 7203 LBM, F-75005, Paris, France
d) CNRS, UMR8640, F-75005, Paris, France
e) Synchrotron SOLEIL Saint-Aubin, 91192, Gif-sur-Yvette Cedex France
f) Laboratoire Friedel, Chimie ParisTech, CNRS, UMR7223, 11, rue Pierre et Marie Curie, 75231 Paris France
g) LCP, CNRS-UMR8000 Université Paris-Sud 11, 91405 Orsay, France
e-mail : [email protected]
Inorganic complexes are increasingly used for biological applications, as metallodrugs or metalloprobes.
Recently, challenging imaging techniques have emerged that led to new information on sub-cellular
distribution of molecules that are important to fully understand their biological activity. The IR-energy
range is particularly attractive for chemical-imaging1 as IR vibrational excitations induce no photobleaching and little photodamage if any. The development of IR-probes is a real challenge in this
emerging field of IR-imaging. Biological media are almost transparent in the range 2200-1800 cm-1.
Metal-CO derivatives show intense absorption bands in this region and have been used since the early
90s as biomarkers.2 Recently, we have shown that Re(CO)3 can be imaged inside cells using a cutting
edge technique coupling an AFM and an IR laser (AFMIR),3 or synchrotron-based IR-light (µ -SRFTIR).4 Interestingly, (L)Re(CO)3 are luminescent when L is a ligand with low-lying π*-orbitals (e.g.
bipyridine). This led us to the idea of designing (L)Re(CO)3 as multi-modal probes for IR and
luminescent bio-imaging that we have called SCoMPI for Single Core Multimodal Probes for
Imaging.5,6 IR-sub-cellular mappings using AFMIR, µ-SR-FTIR, but also Raman and classical
fluorescence will be presented with several examples (hydroxy-tamoxifene hormone, estrogen, long
alkyl chain conjugated with a Re-tris-carbonyl).3,4,5,6 In addition, one of the interest of IR relies on its
easy implementation for direct quantification which will be shown in the talk.7
These SCoMPIs are thus very promising to tag small molecules for imaging and quantification in
biological media.
IR-mapping
Luminescence
References:
1.P. Dumas, N. Jamin, J.-L. Teillaud, L. M. Millerd and B.
Beccarde, Faraday Discuss., 2004, 126, 289.
2. G. Jaouen, A. Vessières, S. Top, A.A. Ismail, I.S. Butler, J.
Am. Chem. Soc. 1985, 107, 4778
3. C. Policar, J. B. Waern, M. A. Plamont, S. Clède, C.
Mayet, R. Prazeres, J.-M. Ortega, A. Vessières and A. Dazzi,
Angew. Chem. Int. Ed., 2011, 50, 860
4. S. Clède, F. Lambert, C. Sandt, Z. Gueroui, N. Delsuc, P.
Dumas, A. Vessières and C. Policar, Biotechnology Advances,
2013, 31, 393
5. S. Clède, F. Lambert, C. Sandt, Z. Gueroui, M.
Réfrégiers, M.-A.Plamont, P. Dumas, A. Vessières, C.
Policar, Chem. Commun., 2012, 48, 7729
6. S. Clède, F. Lambert, C. Sandt, S. Kascakova, M. Unger,
E. Harté, M.-A. Plamont, R. Saint-Fort, A. Deniset-Besseau,
Z. Gueroui, C. Hirschmugl, S. Lecomte, A. Dazzi, A.
Vessières, C. Policar*, Analyst, 2013, 138, 5627
7. S. Clède, F. Lambert, R. Saint-Fort, M.-A. Plamont, H.
Bertrand, A. vessières, C. Policar, Chem. Eur. J., accepted
SCoMPI
13
Superoxide reductase as a model for oxygen activation
S. Rata, S. Ménagea, F. Thomasb, A. Desboisc, V. Nivièrea
a) Laboratoire de Chimie et Biologie des Métaux, UMR 5249, CEA-iRTSV, 17 av. des Martyrs, 38054 Grenoble Cedex 9
b) Département de Chimie Moléculaire, UMR CNRS 5250, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9
c) Laboratoire Stress Oxydant et Détoxication, SB2SM-CNRS URA 2096, iBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette
Cedex
Superoxide reductase (SOR) is a nonheme, iron-containing enzyme, involved in superoxide radical
detoxification O2●- in microorganisms. Its active site consists into an atypical [FeN4S1] square
pyramidal pentacoordinated iron center, where the nitrogen ligands are provided by four histidines and
the sulfur ligand by a cysteine in the axial position (Fig. 1).1,2 Interestingly, SOR presents at least two
striking similarities to cytochrome P450 oxygenase: the presence of Fe3+-OOH intermediates in their
catalytic cycle and the particular coordination [FeN4S1]. The difference in the reactivity between these
two enzymes is essentially due to the evolution of the Fe3+-OOH intermediate. Unlike cytochrome
P450, SOR does not cleave the O-O bond of the Fe3+-OOH unit to generate a high-valent iron-oxo
species, but rather cleaves the Fe-O bond to form its reaction product H2O2.3 Although it was
hypothesized that the differences in the reactivity between SOR and cytochrome P450 could be
ascribed to the nature of the equatorial nitrogen ligands (histidines versus porphyrin ring) and to the
spin state of the Fe3+-OOH species, recent studies have suggested that the residues present in the
second coordination sphere could play more decisive role.
SOR from D. baarsii
Figure 1
Here, we have demonstrated that mutations of different residues of the second coordination sphere can
specifically generate in the SOR active site either a Fe3+-OOH or a Fe=O species, upon reaction with
H2O2. Characterization by Resonance Raman spectroscopy of the SOR mutants4 and studies on their
reactivity as catalysts in oxidation reactions will be presented.
This work illustrates the fact that SOR can be used as an unprecedented model to study the
mechanisms of oxygen activation in metalloenzymes.
1. Lombard M., Touati D., Fontecave M., Nivière V. J. Biol. Chem. 2000, 275, 27021
2. Bonnot F., Molle T., Ménage S., Moreau Y., Duval S., Favaudon V., Houée-Levin C., Nivière V. J. Am. Chem. Soc. 2012,
134, 5120-5130
3.Katona G., Carpentier P., Nivière V., Amara P., Adam V., Ohana J., Tsanov N., Bourgeois D. Science, 2007, 316, 449
4. Bonnot F., Tremey E., Von Stetten D., Rat S., Duval S., Carpentier P., Clemancey M., Desbois A., Nivière V. 2014,
Angewandte Chem. Int. Ed. In press.
14
Efficient Oxidation and Destabilization of Zinc Fingers
by Singlet Oxygen
V. Lebruna, A. Tronb, L. Scarpantoniob, C. Lebrunc, J-L. Ravanatd, J-M. Latoura, N. McClenaghanb and
O. Sénèquea
a) PMB/LCBM/iRTSV, Univ. Grenoble Alpes/CNRS/CEA, 38054 Grenoble
b) ISM, Univ. Bordeaux/CNRS, 33405 Talence Cedex
c) RICC/SCIB/iNAC, CEA, 38054 Grenoble
d) LAN/SCIB/iNAC, CEA, 38054 Grenoble
Singlet oxygen (1O2), the lowest excited state of molecular oxygen, is one of the most reactive
ROS. Despite its occurrence in all aerobic organisms and its ability to severely damage nucleic acids and
proteins[1], its biological chemistry has been neglected in non-photosynthetic organisms. In addition, it
has been demonstrated that living organisms can mount a defense system against 1O2, suggesting a
specific detection pathway. Yet, the cellular and molecular mechanisms involved in this detection
remains poorly understood. Cysteine plays a central role in redox biology, and it is known to react
rapidly with singlet oxygen, yielding several oxidation products (disulfides, thiosulfinates, sulfinates,
sulfonates).[2] However, in many proteins, the sulfur atom of cysteine is bound to a metal, modulating
its reactivity, as exemplified by zinc fingers. Found in a large number of proteins through the entire
living world, these sites contains two, three or four cysteines bound to a ZnII ion, providing structural
stabilization essential for its folding.[3] Nevertheless, rising examples of reactive zinc fingers led to the
emergence of the hypothesis of a new role for them: oxidative stress sensors, via a redox switch
mechanism.[4,5] Because the literature related to the reactivity of zinc fingers toward singlet oxygen is
extremely scarce, we have studied the interaction of 1O2 and zinc finger model peptides to obtain
accurate chemical measurements relevant to biological molecules.
We will report on the reactivity of zinc finger models toward 1O2. Measurement of the reaction
rate and identification of the oxidation products allow us discussing the possibility that such oxidation
occurs in cells and their consequence on zinc finger’s stability.
1.
2.
3.
4.
5.
M.J. Davies Biochimica et Biophysica Acta, 2005, 1703, 93-109.
T.P.A. Devasagayam, A.R. Sundquist, P. Di Mascio; S. Kaiser; H. Sies (1991) J. Photochem. Photobiol. B, 1991, 9, 105–106.
C. Andreini, L. Banci, I. Bertini; A. Rosato, J. Proteome Res., 2006, 5, 196.
K-D. Kröncke ; L-O. Klotz Antioxidants & redox signaling, 2009, 11, 1015-1027
M. Ilbert, P.C.F. Graf, U. Jakob Antioxidants & redox signaling, 2006, 8, 835-846.
15
Sodium Selenite Induced an Apoptotic Cell Death Process and
Exhibited Anti-Tumorigenic Properties in Glioblastoma Cells:
Monolayer (2D) and Multicellular Spheroid Cultures (3D)
Comparisons
F. Hazane-Puch1, J. Arnaud1,2, C. Trocmé3, E. Col5, S. Vergnaud3, L. David-Boudet5, P. Faure1,4
F. Laporte,1 P. Champelovier5
1 UM Biochimie Hormonale et Nutritionnelle (BHN)
2 Inserm, U 1055, Grenoble, FR-38000
3 UM de Biochimie des Enzymes et des Protéines (BEP)
4 Inserm, U 1042, Grenoble, FR 38000
5 Département d’Anatomie et de Cytologie Pathologiques
1, 3 and 5 from: Institut de Biologie et de Pathologie, Centre Hospitalier Universitaire de Grenoble, Hôpital A. Michallon,
CS10217, 38043 Grenoble cedex 09, France.
Selenium (Se), in the form of sodium selenite (Na2SeO3), can modulate cell proliferation, invasion and
cell death in brain tumour cells in monolayer culture (2D culture). However, little is known about its
effects in brain tumour cells (BTC) cultivated in multicellular tumour spheroids (3D culture). Our aim
was to compare the effects of sodium selenite in BTC in 2D or 3D cultures.
Spheroids from LN229, T98G and U87 glioblastoma cell lines were prepared 72h before sodium
selenite supplementation (2.5 µM to 10 µM and 24h to 96h) to evaluate toxicity (MTT, spheroids
diameter), absorption (ICP-MS), cell death (flow cytometry, caspase-3 activity), tumorigenicity
(invasion, gelatin zymography, anhydrase carbonic–IX expression (CA-IX)) and proliferation (flow
cytometry).
Inhibitory concentration 50 (IC50) were comparable in LN229 and U87 (about 4µM and 11 µM in 2D
and 3D respectively) whereas it was higher in T98G. In the 3D, cell lines were about 3 more resistant
than in 2D. The spheroid diameter decreased from 2.5 µM to 10 µM in all BTC. In both 2D and 3D,
sodium selenite absorption was low <1.5% into the BTC lysates, around the IC50. The total Se
recovery in BTC decreased as a function of Se concentration (around the IC50, recovery was 85% in
LN229, 91% in T98G and 84% in U87), suggesting Se volatility: Sodium selenite inhibited cell
proliferation blocking cells in the G2 phase of the cell cycle. Sodium selenite, at micromolar
concentration, induced an apoptotic process dose-, time- and caspase-3-dependent. Autophagy was not
detected: The cell proliferation/invasion process from spheroids was evaluated from 24h to 96h and it
was significantly reduced from 2.5µM 10 µM in the three cell lines. Gelatin zymographies showed that
MMP2 activities were inhibited by sodium selenite more consistently in 2D than in 3D (72h, 2.5 and 5
µM). CA-IX which is highly over-expressed in glioblastoma was not detectable in 2D culture. In 3D, its
expression was decreased by sodium selenite in all BTC (48h, 5 µM).
Although Se was fewly absorbed in glioblastoma tumour cells it induced an apoptotic process via
caspase-3 and inhibited tumorigenicity via proliferation and CA-IX decreases. Since CAIX expression
seems a promising target for human cancer treatment, this 3D model is of interest for studying the
effect of inorganic elements on CA-IX.
16
New homo- and heteronuclear complexes of Manganese/Calcium:
spectroscopic models for the investigation of calcium in the OxygenEvolving Center of Photosystem II
B. Gereya, M. Gennaria, E. Gouréa, J. Pécautb, V. Martin-Diaconescuc, J. Fortagea, S. DeBeerc, F. Neesec,
C. Duboc,a M.-N. Collomba
a) Chimie Inorganique Redox, DCM, Université Joseph Fourier
b) Reconnaissance Ionique et Chimie de Coordination, SCIB, INAC, CEA Grenoble
c) Max-Planck-Institute for Chemical Energy Conversion
The active site of the photosystem II (PSII) also called the Oxygen-Evolving-Center (OEC) – an
inorganic oxygen-bridged cluster of four manganese and one calcium ions (Mn4CaO5) stabilized by
carboxylate ligands – catalyzes the four electron oxidation of water into dioxygen1. The recent crystal
structure of PSII at the 1.9 Å resolution2 evidences for the first time the presence of four “water”
molecules (of unknown protonated states) directly bound to the cluster (two coordinated to the Ca and
the other two to one Mn in close proximity), that could act as substrates for dioxygen formation.
However, the mechanism of water oxidation at the molecular level, including the O-O bond formation,
and the role(s) of Ca, are still far from being elucidated. To date, less than fifteen examples of synthetic
hetero-nuclear manganese-calcium complexes have been isolated and only two examples reproduce the
cubane unit of the OEC3,4.
In this context, we develop a new family of homonuclear calcium complexes as well as heteronuclear
MnII/Ca complexes incorporating aquo ligands, as found in the OEC. To access these complexes,
tripodal ligands including the pyridine-carboxylate have been synthesized. Figure 1 displays the X-Ray
structures of two selected complexes. We also deeply investigate their electronic structure by state-ofthe-art Mn and Ca X-Ray absorption (XAS) and emission (XES) techniques, combined to theoretical
chemistry in collaboration with the groups of Profs S. DeBeer and F. Neese of the Max Planck
Institute of Mülheim,. Calcium K-Edge XAS and XES spectroscopies remain largely unexplored and
the investigation of well-characterized model Ca complexes will provide essential insights for the
understanding of more complex systems such as the Mn4Ca cluster of the OEC. Preliminary results
obtained by XAS Ca spectroscopy will be also presented.
[MnII(tpada)ClCa(OH2)3(MeOH)2]Cl
[Ca(tpaaH)(OH2)]•3H2O
1.
2.
3.
4.
B. Kok, B. Forbush, M. McGloin, Photochem. Photobiol., 1970, 11, 457
Y. Umena, K. Kawakami, J.-R. Shen, N. Kamiya, Nature, 2011, 473, 55
J. S. Kanady, E. Y. Tsui, M. W. Day, T. Agapie, Science, 2011, 333, 733
S. Mukherjee, J. A. Stull, J. Yano, T. C. Stamatatos, K. Pringouri, T. A. Stich, K. A. Abboud, R. David Britt, V. K.
Yachandra, G. Christou, Proc. Nat. Ac. Sci., 2012, 109, 2257
17
NAD biosynthesis in procaryotes: Structural and functional studies of
the Quinolinate Synthetase
A. Chana, D. Reichmanna, M. Clémanceya, M. Cherrierb, C. Darnaultb, O. Hamelina, J.-M. Latoura,
P. Amaraa, J. C. Fontecilla-Campsb, J.-M. Mouescac, S. Ollagnier de Choudensa
a) Laboratoire de Chimie et Biologie des Métaux, iRTSV/LCBM, UMR5249, 17 Avenue des Martyrs, 38054 Grenoble
Cedex 09, France.
b) Laboratoire des Résonances Magnétique, DSM/INAC/SCIB, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France.
c) Groupe Métalloprotéines, Institut de Biologie Structurale, 71 avenue des Martyrs, 38044 Grenoble Cedex 9
email : [email protected]
The Nicotinamide Adenine Dinucleotide (NAD) is a key cofactor essential for cellular
metabolism. It is synthesized from quinolinic acid (QA) in all living organisms, the biosynthesis of
which is different between eucaryotes and procaryotes. Indeed, most of eukaryotes produce QA from
L-tryptophan, whereas most of prokaryotes and plants synthesize QA by the concerted action of 2
enzymes: L-aspartate oxydase (NadB), an FAD enzyme, which catalyzes L-Aspartate oxidation to form
iminoaspartate (IA) while quinolinate synthetase (NadA) allows condensation between IA and
Dihydroxyacetone Phosphate (DHAP) to produce QA.Besides this « de novo » pathway, most
eukaryotes and some bacteriae have a salvage pathway which allows NAD synthesis from metabolites
of NAD degradation or cell nucleotides in order to maintain a correct pool of NAD in the cell 1.
However, some pathogens like Mycobacterium leprae and Helicobacter pylori do not possess this pathway2.
As a consequence, NadA represents a very attractive target for the design of specific antibacterial
agents since it does not exist in human.
Quinolinate synthetases from different organisms were characterized and contain all a [4Fe2+
4S] cluster essential for activity3-5. However, the role of the cluster in catalysis is not known.
Nevertheless, the fact that it is coordinated by only three cysteine ligands indicates that it may play a
Lewis acid role in the reaction like for aconitase or others dehydratases clusters. Besides, the molecular
mechanism of quinolinic acid formation from IA and DHAP is not known as well. Therefore, during
my phD, we aimed at:
- Elucidating the molecular mechanism of the reaction catalysed by NadA using substrates or
intermediates analogs
- Demonstrating cluster role in catalysis using spectroscopic approaches.
These two points will be illustrated by the use of an intermediate analog : 4, 5dithiohydroxyphthalic acid (DTHPA) which was studied using biological, biochemical &
spectroscopic methods.
- Getting NadA tridimensional structure in order to understand better NadA mechanism.
Structure resolved at 1.6Ǻ of NadA from Thermotoga maritima with its 4Fe-4S cluster will be
presented with some insights into NadA mechanism by our collaborator J. FontecillaCamps.
- Finding some inhibitory molecules against NadA from M.leprae and H.pylori. These
molecules may be used as antibacterial agents.
This point will not be presented during the talk but will be discussed in a poster. (D. Reichmann)
1. Begley T.P., Kinsland C., Mehl R.A., Osterman A., Dorrestein P. Vitam. Horm. 2001, 61, 103-19 (2001).
2. S.Y. Gerdes. J Bacteriol 2002, 184, 4555-4572.
3.Ollagnier-de Choudens, S., Loiseau, L., Sanakis, Y., Barras, F. & Fontecave, M. FEBS Lett 2005, 579, 3737-3743.
4. Rousset, C., Fontecave, M. & Ollagnier de Choudens, S., FEBS Lett 2008, 582, 2937-2944.
5. Saunders, A.H. & Booker, S.J. Biochemistry 2008, 47, 8467-8469.
6. Chan, A, Clémancey M, Mouesca JM, Amara P, Hamelin O, Latour JM & Ollagnier de Choudens S., Angew Chem Int Ed
Engl. 2012 Jul 27; 51(31):7711-4.
18
The crystal structure of FeS quinolinate synthase unravels an
enzymatic dehydration mechanism that uses tyrosine and a hydrolasetype triad
M. V. Cherriera, A. Chanb, C. Darnaulta, D. Reichmannb, P. Amaraa, S. Ollagnier de Choudensb,
J. C. Fontecilla-Campsa
a) Metalloproteins Unit, Institut de Biologie Structurale, Commissariat à l’Energie Atomique–Centre National de la Recherche
Scientifique–Université Grenoble-Alpes, 71, Av. des Martyrs, CS 10090, 38044 Grenoble, Cedex 09,France
b) DSV/iRTSV/CBM, UMR 5249 CEA-Université Grenoble I-CNRS/Equipe Biocatalyse, CEA-Grenoble, 17 Rue des
Martyrs, 38054 Grenoble Cedex 09, France
Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis
of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide
(NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one
substrate analog, providing partial mechanistic information. At the meeting we will report on the
holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a
cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only
suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a
conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases1. Our
mutagenesis data show that all these residues are essential for activity and strongly suggest that Tyr21
deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA
displays a dehydration mechanism significantly different from the one found in archetypical
dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The Xray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of
NAD biosynthesis.
1.
Cherrier et al., J. Amer. Chem. Soc. 2014, 136, 5253-5256.
19
Thermodynamics + redox couples + metals = Life
W. Nitschke
BIP (UMR 7281)/CNRS, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20
The 2nd law of thermodynamics demands that the extraordinary decrease in entropy of the system called
“life” must be counterbalanced by a more than compensating dissipation of some sort of energy. In
basically all forms of life on planet Earth, this energy is redox disequilibrium, that is, the simultaneous
presence of lower potential reduced and higher potential oxidised redox compounds, a quintessential
out-of-equilibrium situation (note that photosynthesis doesn’t contradict this principle since only the
disequilibrium situation is induced by life but the free energy converting mechanisms are the same as in
non-photosynthetic systems). Bioenergetics is the field studying life’s myriad ways of collapsing all
kinds of redox disequilibria available in the habitable zone of our planet. Bioenergetic mechanisms are
predominantly, although not exclusively, based on metalloenzymes as redox catalysts. I will try to distill
a number of universal thermodynamic/electrochemical principles from the plethora of bioenergetic
free energy converting systems.
20
N2O reduction at a mixed-valent {Cu2S} center
C. Esmieua, M. Oriob, L. Le Papea, C. Lebrunc, J. Pécautc, S. Torelli,a S. Ménagea
a) Laboratoire de Chimie et Biologie des métaux. UMR 5249 Université Grenoble Alpes – Grenoble France, CNRS et CEA –
CEA DSV-iRTSV - 17, Avenue des Martyrs, 38054 Grenoble
b) Université des Sciences et Technologies de Lille - Laboratoire de Spectrochimie Infrarouge et Raman - Bâtiment C5 - UMR
CNRS 8516 - 59655 Villeneuve d'Ascq Cedex
c) Laboratoire de Reconnaissance Ionique et Chimie de Coordination – UMR E3 Université Joseph Fourier et CEA SCIB/INAC –CEA Grenoble–17, Avenue des Martyrs, 38054 Grenoble
Since twenty years, environmental impacts of Nitrous oxide (N2O) are known (Kyoto
protocol). N2O is indeed a potent greenhouse agent and a dominant ozone depleting molecule.[1] It is
released by biological processes like in bacterial respiration, nitrification and denitrification pathways.
However it is also produced by the industry and its rising atmospheric concentration is mainly due to
anthropogenic activities. In recognition of these facts, the need of remediation processes has stimulated
a lot of interest, particularly through the use of transition metal catalysts. Moreover, in Nature, one
metalloenzyme (N2O-reductase) cleanly converts N2O in N2 via a unique tetranuclear µ4-sulfido copper
center named Cuz.[2] A mechanism for N2O reduction has been proposed and involves an N2O binding
between two of the four copper ions.[3] Today, there is no active system that degradates this gas under
mild conditions.
Following a bioinspired approach related to the previous hypothesis, our team is seeking to reproduce
CuZ reactivity. Herein, we report the synthesis of novel binuclear copper complexes capable of N2O
reduction. These compounds, synthetized via an original method, contain a thiophenolate ligand with
N-coordinating atoms.[4] These new complexes have been spectroscopically and theoretically
characterized, and present a {Cu2S}2+ mixed-valent motif where the delocalization depends on the
organic ligand. The reactivity toward N2O depends of the presence of a labile site, which potentially
indicates the coordination of this weak substrate. In a biomimetic approach, we are also able to
synthetize a new tetranuclear copper (II) complex with the same starting ligand which presents a new
kind of reactivity versus O2.
[1] Ravishankara, A. R., et al., Science, 2009, 326, 123-125.
[2] Einsle, O., and coll., Biol. Chem. 2012, 393, 1067–1077.
[3] Solomon, E. I., and coll., J. Am. Chem. Soc. 2007, 129, 3955-3965.
[4] Torelli, S., and coll., Angew. Chem., Int. Ed. 2010, 49, 8249-8252.
21
Visible light hydrogen production in water from a cobalt(III) tetraazamacrocyclic catalyst
C. E. Castillo,1 S. Varma,1 T. Stoll, 1 J. Fortage,1 A. G. Blackman,1 F. Molton,1 A. Deronzier,1
M.-N. Collomb.1
1) Université Joseph Fourier Grenoble 1/CNRS, Dèpartement de Chimie Moléculaire, UMR 5250, Institut de Chimie
Moléculaire de Grenoble, FR-CNRS-2607, Laboratoire de Chimie Inorganique Rédox, BP 53 – 38041 Grenoble cedex 9 ;
France.
Solar light-induced water splitting into hydrogen (H2) and oxygen, also referred as artificial
photosynthesis, is a very attractive sustainable approach to produce H2, one of the most promising
clean and renewable fuels for the future.1 During the past decade, numerous efficient molecular
homogeneous photocatalytic systems have been reported and they combined a photosensitizer (PS), a
hydrogen evolving catalyst (HEC), and a sacrificial electron donor. Most of these systems operate in
organic or mixed aqueous-organic solvents and only a few of them are active in fully aqueous solution
usually due to the poor stability of the catalyst in water.
We recently published a very efficient three-component
homogeneous system
for visible-light-driven H2
production in aqueous solution based on
[RhIII(dmbpy)2Cl2]+ (Rh) as the HEC, [Ru(bpy)3]2+ as
the PS, and ascorbic acid/sodium ascorbate as the
sacrificial electron donor (Scheme 1).2 The catalytic
performance of this system was also significantly
improved by the covalent linkage of the Cat and the PS
resulting in the first efficient single-component
molecular photocatalyst (Ru2Rh) for H2 production in
pure aqueous solution.3
In this communication we will present our results on a
similar system, a tetraaza-macrocyclic catalyst based on
cobalt [Co(CR)Cl2]2+, a more earth-abundant metal.
Comparative studies in aqueous solution with some of
the most efficient cobaloxime and rhodium catalysts
reported in water, show that the new cobal H2-evolving
catalyst is by far the most active homogeneous
photocatalytic system for H2 production.4
1.
M. J. Esswein, D. G. Nocera, Chem. Rev. 2007, 107, 4022.
2.
T. Stoll, M. Gennari, I. Serrano, J. Fortage, J. Chauvin, F. Odobel, M. Rebarz, O. Poizat, M. Sliwa, A. Deronzier, M.-N.
Collomb, Chem. Eur. J. 2013, 19, 782.
3.
T. Stoll, M. Gennari, J. Fortage, C.E. Castillo, M. Rebarz, M. Sliwa, O. Poizat, F. Odobel, A. Deronzier, M.-N.
Collomb, Angew. Chem. Int. Ed. 2014
4.
S. Varma, C. E. Castillo, T. Stoll, J. Fortage, A. Blackman, F. Molton, A. Deronzier, M.-N. Collomb, Phys. Chem. Chem.
Phys. 2013, 15, 17544.
22
Transmembrane signal transduction by the CnrYXH complex in
Cupriavidus metallidurans CH34: interaction between CnrX and CnrY
W. Ziani, A. Maillard, I. Petit-Haertlein, E. de Rosny, J. Covès
Institut de Biologie Structurale, 6 rue J. Horowitz, 38000 Grenoble.
The CnrYXH complex contributes to regulate the expression of the genes involved in cobalt and nickel
resistance in Cupriavidus metallidurans CH34. CnrX is a membrane-anchored protein with a metal-sensor
domain protruding in the periplasm 1, 2, 3. CnrX interacts with the bitopic antisigma factor CnrY that
sequesters the ExtraCytoplasmic Function (ECF) sigma factor CnrH on its cytoplasmic extremity. The
binding of Ni(II) or Co(II) to CnrX in the periplasm triggers the signal transduction that leads to
release CnrH into the cytoplasm which is required to initiate the transcription of the genes involved in
resistance. The nature of the signal passed through the membrane is still unknown. The first step of
this transduction is the sensing of nickel and cobalt by the periplasmic domain of CnrX (CnrXs).
Structures of CnrXs under the Ni-, Co-, and Zn-bound forms have shown how Zn-to-Ni or Zn-to-Co
substitution in the metal binding site imposes a switch in coordination geometry and induces
conformational changes affecting CnrXs dimer.
What are the next steps following this sensing and leading to the release of CnrH in the cytoplasm? In
this talk, we will show that CnrX interacts with CnrY in the periplasm, thus suggesting that signal
transduction could proceed through the modulation of CnrX:CnrY interaction upon Ni(II) or Co(II)
binding to CnrX periplasmic domain.
1. Trepreau, J. et al., Journal of Molecular Biology, 2011, 408, 766-779.
2. Trepreau, J. et al., Biochemistry, 2011, 50, 9036-9045.
3. Trepreau, J. et al., Metallomics, 2014, 6, 263-273.
23
Cu and Zn impacts in Alzheimer's disease
C. Hureaua,b, E. Grasa,b, P. Fallera,b
a) CNRS; Laboratoire de Chimie de Coordination, Toulouse, (France).
b) Université de Toulouse, UPS, INPT; LCC; Toulouse, (France).
Alzheimer's disease (AD) is characterized by a global deterioration of mental, cognitive and physical
abilities. AD is the most common cause of dementia in the elderly population. As a direct consequence,
AD represents a global public health problem that will become even more important in the next years.
Two hallmarks are detected in AD patients brains: i) the intracellular neurofibrillary tangles of Tau
protein, the protein involved in the "neuronal skeleton" and ii) the extracellular amyloid plaques (also
known as senile plaques) made of aggregated forms of the amyloid-β (Aβ) peptide. These peptides are
present in soluble (monomeric) form in healthy brains. Hence the route to the formation of the
aggregated forms of the Aβ peptide is key in the etiology of the disease. Such phenomenon is known as
the amyloid cascade (Figure). A role in the amyloid cascade have been proposed for Copper(I/II) and
Zinc(II). They have also been implicated in oxidative stress production, i.e. in the formation of highly
toxic reactive oxygen species (ROS).
In the Biological Chemistry Group, we have recently studied how such metallic ions are bound to the
Aβ peptide, which is a prerequisite to understand how they can interfere in the Aβ aggregation process
as well as in the ROS production. In particular, we have shown how the nature of the metallic ion and
the peptide sequence impact the coordination site.
The control of these data are crucial for designing new kind of therapeutic tools, which is an emergent
axis of our team. Some of these terapeutic strategies will be briefly commented on.
1.
C. Hureau, Coord. Chem. Rev. 2012, 256, 2164.
2.
C. Hureau, Act. Chim. 2013, 380, 31.
24
Metallobiology of host-pathogen interactions:
an intoxicating new insight
O. Neyrollesa
a) Centre National de la Recherche Scientifique & Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et
de Biologie Structurale, Toulouse, France
Iron, zinc and copper, among others, are transition metals with multiple biological roles that make
them essential elements for life. Beyond the strict requirement of transition metals by the vertebrate
immune system for its proper functioning, novel mechanisms involving direct metal intoxication of
microorganisms are starting to be unveiled as important components of the immune system, in
particular against Mycobacterium tuberculosis. In parallel, metal detoxification systems in bacteria have
been recently characterized as crucial microbial virulence determinants (1-5). Here, I will focus on these
exciting advancements implicating copper- and zinc-mediated microbial poisoning as a novel innate
immune mechanism against microbial pathogens, shedding light on an emerging field in the
metallobiology of host-pathogen interactions.
1.
2.
3.
4.
5.
Botella H, Stadthagen G, Lugo-Villarino G, de Chastellier C, Neyrolles O. Metallobiology of host-pathogen
interactions: an intoxicating new insight. Trends Microbiol 2012 20(3):106-12
Botella H, Peyron P, Levillain F, Poincloux R, Poquet Y, Brandli I, Wang C, Tailleux L, Tilleul S, Charrière GM,
Waddell SJ, Foti M, Lugo-Villarino G, Gao Q, Maridonneau-Parini I, Butcher PD, Castagnoli PR, Gicquel B, de
Chastellier C, Neyrolles O. Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human
macrophages. Cell Host Microbe 2011 10(3):248-59
Wolschendorf F, Ackart D, Shrestha TB, Hascall-Dove L, Nolan S, Lamichhane G, Wang Y, Bossmann SH, Basaraba
RJ, Niederweis M. Copper resistance is essential for virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A
2011 108(4):1621-6
Ward SK, Abomoelak B, Hoye EA, Steinberg H, Talaat AM. CtpV: a putative copper exporter required for full
virulence of Mycobacterium tuberculosis. Mol Microbiol 2010 77(5):1096-110
Hood MI, Skaar EP. Nutritional immunity: transition metals at the pathogen-host interface. Nat Rev Microbiol 2012
10(8):525-37
25
Biochemical characterization of AtHMA8/PAA2, a chloroplastthylakoid Cu(I)-ATPase
E. Sautron,a P. Catty,b D. Pro,a N. Rolland,a D. Seigneurin-Bernya
a) Laboratoire Physiologie Cellulaire et Végétale UMR5168-CNRS/CEA-DSV iRTSV/USC1359-INRA/ UJF Grenoble
b) Laboratoire Chimie et Biologie des Métaux UMR5249-CNRS/CEA-DSV iRTSV/UJF Grenoble
17 rue des Martyrs -38054 Grenoble, France
email : [email protected]
Copper is a transition metal essential for plant development which acts as structural or catalytic element
in proteins. Thanks to his redox properties copper is involved in many biological processes. In
Arabidopsis chloroplast, copper is an essential cofactor required for superoxide radicals’ detoxification
(via Cu/Zn superoxide dismutase) and photosynthetic electron transfer (via plastocyanin). However, its
chemical properties make copper toxic when present in excess in the cell. To prevent copper toxicity,
cells throughout evolution have evolved a finely tuned homeostasis specific of this metal ion.
Membrane transport proteins, among them PIB-type ATPases, play crucial role in the regulation of
copper concentration, In Arabidopsis thaliana, three PIB-type ATPases are involved in chloroplast copper
homeostasis: AtHMA11 and HMA62 at the envelope membrane and HMA83 at the thylakoids
membrane. So far, only HMA6 has been biochemically characterized4. The role of HMA8 has only
been deduced from reverse genetic analyses in planta.
We have carried out the biochemical study of HMA8. Native and mutated forms of the transporter
have been produced in Lactoccoccus lactis. The enzymatic properties have been determined by
phosphorylation assays in vitro, and confirmed by phenotypic analysis in yeast. This study shows that
HMA8 is a high affinity copper(I) transporter, with enzymatic properties distinct from those of HMA6.
1. D. Seigneurin-Berny, A. Gravot, P. Auroy, C. Mazard, A. Kraut, G. Finazzi, F. Rappaport, A. Vavasseur, J. Joyard, P.
Richaud, N. Rolland, Journal of Biological Chemistry, 2006, 281, 2882-2892.
2. T. Shikanai, P. Müller-Moulé, Y. Munekage, K.K. Niyogi, M. Pilon, Plant Cell, 2003, 15, 1333-1346.
3. S.E. Abdel-Ghany, P. Müller-Moulé, K.K. Niyogi, M. Pilon, T. Shikanai, Plant Cell 2005, 17, 1233-1251.
4. P. Catty, S. Boutigny, R. Miras, J. Joyard, N. Rolland, D. Seigneurin-Berny, Journal of Biological Chemistry, 2011, 286, 3618836197.
26
Artificial inorganic nucleases involving the phenolate-copper entity
D. Serre,a N. Berthet,b V. Frachet,c C. Philouze,a X. Ronot,c F. Thomasa
a) Département de Chimie Moléculaire, Chimie Inorganique Redox (CIRE), UMR CNRS 5250, Université J. Fourier, B.P. 53,
38041 Grenoble cedex 9, France.
b) Département de Chimie Moléculaire, Ingénierie et Interactions Biomoléculaires (I2BM), UMR CNRS 5250, Université J.
Fourier, B.P. 53, 38041 Grenoble cedex 9, France.
c) Age – Imagerie – Modélisation, Cancer Cycle cellulaire et Sénescence (CaCyS), Université Joseph Fourier, FRE 3405, EPHE,
38706 La Tronche cedex, France.
Copper complexes based on N3O tripodal ligands (two pyridines, one tertiary amine and one phenol
donors) are known to be good structural models of the active site of the enzyme galactose oxidase
(under its inactive form).1 The phenol could be oxidized into phenoxyl radical, mimicking the enzyme
under its active form, potent for the oxidation of primary alcohols into aldehydes. We recently showed
that some of these complexes could also exhibit a significant DNA cleavage activity.2 This nuclease
activity is however observed only if at least one pyridine is methylated in a-position. This might be
explained by a lowering of the phenol’s pKa promoted by a geometrical rearrangement (axial/equatorial
isomerization of the substituted pyridine/phenolate). The fact that the nuclease activity is systematically
observed whatever is the phenol substituent (F, OMe) suggests that phenoxyl radical generation is not
directly implied in the process. This important observation made us envision multiple
functionalizations on the phenol, which will be presented, in order to enhance the biological activity of
the compounds.
The incorporation of cationic & intercalating sub-units improved the nuclease activity by a factor 100.
In addition, a significant anti-proliferative activity was observed against cancer cells at micromolar
concentrations.
O
R
O
N
Cu
N
O
N
N
Cu
N
H2O
H 2O
(a)
(b)
N
Figure 1. Artificial copper nucleases: (a) = from reference 2; (b) = this work.
1.
F. Thomas, Eur. J. Inorg. Chem. 2007, 2379.
2.
N. Berthet, V. Martel-Frachet, F. Michel, C. Philouze, S. Hamman, X. Ronot, F. Thomas, Dalton Trans. 2013, 42, 8468.
27
Photocatalytic Oxidation of Organic Substrates by Ru/Cu Heterometallic Complexes with Dioxygen as the Unique Oxygen Atom
Source
W. Iali a, O. Hamelin,a S. Torelli,a S. Menage,a P. H. Lanoe,b D. Jouvenot,b F. Loiseaub
a) Laboratoire de Chimie et Biologie des Métaux, UMR 5249-Université Grenoble, I-CNRS-CEA, CEA Grenoble, 17
Avenue des Martyrs, 38054 Grenoble, France.
b) DCM-Equipe CIRE- Université de Grenoble, 38400 Saint-Martin-d’Hères
Currently, processes using atmospheric dioxygen in fine chemistry for oxidation reactions are limited
mainly due to the difficulty to activate such a stable molecule. However, some Fe(II) and Cu(I)enzymes i.e. oxygenase are capable to realize this activation yielding to oxidizing species. After a two
electron oxidation of a substrate, Cu(I) and Fe(II) are recovered and their subsequent reduction are
then required to regenerate the reduced active species.
With the aim to develop new “eco-aware” catalysts we designed an original heterobinuclerar Ru-Cu
complex constituted by the covalent association of a Ru-based photosensitizer and a Cu-based catalyst.
Irradiation at the appropriate wavelength, in the presence of a sacrificial electron-donnor (D), results in
the reduction of the Cu(II) system into Cu(I) thanks to electron transfers (E.T) from the electrondonnor to the copper subunit via the excited photosensitizer. Photocatalytic experiments showed that
the synthesized complex was able to selectively oxidize sulfides into sulfoxides using O2 as the unique
oxygen-atom source. In order to perform oxygenation reactions, the use of dioxygen as an oxygen atom
source appears to be an ideal solution.
D
Sulfure
D+
Sulfoxide
28
Grafting of 3,4-epoxybutyltrimethoxysilane on silicon oxide
by supercritical carbon dioxide
J. Rull,a,b,c,d G. Nonglaton,a G. Costa,a C. Fontelaye,a C. Marchi-Delapierre,b,c,d S. Ménage,b,c,d
G. Marchanda
a) CEA, Leti, MINATEC Campus, 17 rue des Martyrs, F38054 GRENOBLE, Cedex 9, France.
b) CEA, iRTSV, LCBM, 38054 GRENOBLE, France.
c) CNRS, UMR 5249, GRENOBLE, France.
d) Université Joseph Fourier Grenoble I, GRENOBLE, France.
It is well known that high quality organosilane monolayers are difficult to form using the conventional
solution-phase methodology [1]. Our research probes the surface modification addressed through the
silanization of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon wafers using supercritical carbon
dioxide (scCO2) as a solvent. The best supercritical conditions were established and unequivocally
confirmed by fluorescence and Surface Enhanced Ellipsometric Contrast optical technique. The
silanization performance of the supercritical fluid deposition was compared with traditional solution
and molecular vapor deposition methods, evidencing that supercritical CO2 offers many advantages
instead liquid and vapor phase, homogeneity, less reaction time, among all the eco-aware properties of
the scCO2 [2, 3]. The synthetized organosilane monolayers were completely characterized by contact
angle, multiple internal reflection, atomic force microscopy and X-ray photoelectron spectroscopy
(XPS). The grafting efficiency of amino-modified molecule on epoxy functionalized surface was
evaluated by fluorescence and XPS. This study represents the first step for the formation of an
EBTMOS solid scaffold using scCO2, which has a broad range of applications such as introducing
interesting organic, inorganic and bioorganic molecules for several applications [4]. Preliminary studies
of the scope of its applications have been done and work is in progress to extrapolate this scCO2
grafting conditions to silica beads and carbon nanotubes.
1. A. Y. Fadeev, T. J. McCarthy Langmuir 1999, 15, 7238−7243.
1. D. Rébiscoul, V. Perrut, O. Renault, F. Rieutord, S. Olivier, P.-H. Haumesser J. Supercrit. Fluid. 2009, 51, 287–294.
2. C. Domingo, E. Loste, J. Fraile J. Supercrit. Fluid. 2006, 37, 72–86.
3. E. Grinenval, G. Nonglaton, F. Vinet Appl. Surf. Sci. 2014, 289 (0), 571−580.
4. Manuscript submitted.
29
Structural insights into a novel radical based carbon-sulfur bond
formation in radical SAM enzyme HydE from Thermotoga maritima
R. Rohac, A. Benjdia, O. Berteau, P. Amara, J. C. Fontecilla-Camps, Y. Nicolet
Métalloprotéines, Institut de Biologie Structurale J.P. Ebel, 71 avenue des Martyrs, CS 10090, 38044 Grenoble, France
Radical SAM enzymes correspond to a growing superfamily of iron-sulfur containing proteins that
usually proceed through the reductive homolytic cleavage of S-adenosyl methionine (SAM) to produce
L-methionine and a 5’-deoxyadenosyl radical (5’-Ado·). The latter can subsequently abstract an H-atom
from an unfunctionalized carbon and thus catalyze many different reactions. Among these reactions is
the formation of carbon-sulfur bonds as performed by, for example BioB, MiaB, RimO or AlbA. All
these enzymes generate 5’-Ado· from SAM with the help of a reduced Fe4S4 cluster and subsequently
produce a radical on one of the substrate’s carbon atoms. Furthermore, they use an additional FeS
cluster to either donate a S2- ion or activate a target thiolate ion.
Using high-resolution structures of the radical SAM FeFe-hydrogenase maturase HydE, we
were able to follow the reaction of thioether bond formation involving a direct carbon-centered radical
attack on a thioether sulfur atom, without the requirement of an FeS cluster as either sulfur donor or
activator. These results shed considerable light on the mechanism involved in radical C-S bond
formation, a process with foreseeable applications in chemical synthesis.
30
Water splitting: from bio-inspiration to system integration
N. Kaeffera, M. Fournierb, D. Méndez-Hernándezb, M. Tejedab, E. Reyesb, J. Tomlinb, M. ChavarotKerlidoua, V. Arteroa, T.A. Mooreb, A. L. Mooreb and D. Gustb
a) Laboratoire de Chimie et Biologie des Métaux (Université Grenoble Alpes/CEA/CNRS), 17 rue des Martyrs, F-38054
Grenoble Cedex 9, FRANCE. www.solhycat.com
b) Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
A key challenge in water-splitting is to implement individual components into true operating devices.
Taking our inspiration from photosynthetic systems where both reductive and oxidative catalytic
processes are coupled along with light-harvesting, we will report here on our ongoing efforts to couple
an O2-evolving photoanode1,2 designed at Arizona State and Pennsylvania State Universities with a Cobased H2-evolving cathode3 developed in our group so as to build a water-splitting cell that operates
under fully aqueous conditions. In our tandem design, supplementary dye-sensitized solar cells
mounted in series within the water-splitting cell provide additional photo-potential, making the cell
independent of external bias.
This collaborative work has been carried out within the framework of a fellow-exchange program
funded by the Solar Fuels Institute (SOFI, www.solar-fuels.org).
1. W. J. Youngblood, S. H. A. Lee, Y. Kobayashi, E. A. Hernandez-Pagan, P. G. Hoertz, T. A. Moore, A. L. Moore, D.
Gust and T. E. Mallouk, J. Am. Chem. Soc., 2009, 131, 926-927.
2. Y. X. Zhao, J. R. Swierk, J. D. Megiatto, B. Sherman, W. J. Youngblood, D. D. Qin, D. M. Lentz, A. L. Moore, T. A.
Moore, D. Gust and T. E. Mallouk, Proc. Natl. Acad. Sci. USA, 2012, 109, 15612-15616.
3. E. S. Andreiadis, P.-A. Jacques, P. D. Tran, A. Leyris, M. Chavarot-Kerlidou, B. Jousselme, M. Matheron, J. Pécaut, S.
Palacin, M. Fontecave and V. Artero, Nat. Chem., 2013, 5, 48-53.
31
A Ru based artificial oxydase
L. Rondot,a A. Jorge Robin,a C. Marchi-Delapierre,a C. Cavazza,a S. Ménagea
a) Laboratoire de Chimie et Biologie des métaux. UMR 5249 Université Joseph Fourier, CNRS et CEA – CEA-Grenoble 17, Avenue des Martyrs, 38054 Grenoble
One of the research activities of the BioCE team concerns the development of catalytic oxidation
reactions that mimic the activity of oxygenases in order to perform oxidation reactions following the
green chemistry principles. The team then invested in the design of new bio-inorganic catalysts:
artificial metalloenzymes. This allows us to find an alternative to polluting solvents (such as water),
toxic and hazardous chemicals.
In this context, we have developed two new biocatalysts with NikA as the protein scaffold. One, an
artificial oxydase, is capable of epoxidation catalysis, in organic media, in the presence of iodobenzene
diacetate. The other, an artificial oxygenase, incorporates a Fe (III) complex is able to activate
molecular oxygen which is the cleanest oxidant and to transfer it to a substrate of interest.
We so have transposed the oxidation reactivity to the corresponding hybrids, affording it milder and
greener condition. Our catalysis results are supported by X-rays analysis on protein crystals.
I will present you the design of the corresponding hybrids and our latest results concerning their in
alkene oxidation catalysis.
32
Original Lanthanide Luminescent bioprobes for new biphotonic
microscopies
O. Maury
Laboratoire de chimie, UMR5182 CNRS – Université de Lyon 1 - ENS Lyon
The sensitization of lanthanide luminescence by nonlinear two-photon (2P) absorption process allows
to combine the intrinsic advantages of rare earth spectroscopy (line shape emission, long lifetime) and
those of biphotonic microscopy (NIR excitation, 3D resolution…) for bio imaging applications.1
Nowadays most studies on two photon lanthanide luminescence bioprobes (2P-LLB) are focused on
Eu(III) for cell imaging applications2 and the fast incident laser repetition rate (10 ns/pulse) was
thought to preclude any two-photon time-resolved microscopy imaging. In this contribution, we
describe a complete series of 2P-LLB based on functionnalised TACN ligands that enable to sensitize
Eu(III), but also Sm, Tb, Dy and Yb emitting in the NIR. The microscopy instrumentation was
developed to allow performing two-photon bio-imaging of thick tissue in an unconventional NIR-toNIR configuration.3 Unprecedented two-photon multiplexing experiments were also performed in the
visible using a combination of Eu and Tb 2P-LLB. In addition, we demonstrate that the long
luminescence lifetime of LLB can be measured and imaged in cells using the conventional confocal 2P
microscope with high accuracy using the new Temporal Sampling Lifetime Imaging Microscopy
(TSLIM) method opening the way to 2P-FRET imaging. Finally 2P-time gated imaging experiments are
reported using a pinhole shifting Lifetime Imaging Microscopy (PSLIM).4
1. For a review see C. Andraud, O. Maury Eur. J. Inorg. Chem. 2009, 4357-4371
2. A. Picot, A. D'Aléo, P.L. Baldeck, A. Grichine, A. Duperray, C. Andraud, O. Maury J Am Chem Soc. 2008,130, 1532-3.
3. A. D'Aléo, A. Bourdolle, S. Brustlein, T. Fauquier, A. Grichine, A. Duperray, P.L. Baldeck, C. Andraud, S. Brasselet, O.
Maury Angew Chem Int Ed. Engl. 2012, 51, 6622-5.
4. A. Grichine, A. Haefele, S. Pascal, A. Duperray, R. Michel, C. Andraud, O. Maury Submitted
33
Lanthanide-based luminescent probe for time-gated detection of
copper(I) : modulation of the antenna effect by cation/̟ interaction
M. Isaac,a A. Roux,a S. A. Denisov,b G. Jonusauskas,b N. D. McClenaghan,b J.-M. Latour,a O. Sénèquea
a) Laboratoire de Chimie et Biologie des Métaux, Equipe Physicochimie des Métaux en Biologie, iRTSV/CEA.
b) Institut des Sciences Moléculaires, CNRS / Univ. Bordeaux
Copper is an essential metal for life but it can also be toxic. Therefore, its homeostasis is tightly
regulated by living systems. Serious diseases like Menkes and Wilson’s disease are linked with a lack or
an excess of copper. Hence it is important to design quantitative copper probes for a better
understanding of its regulation and be able to diagnose diseases.
In cells, copper is at the oxidation state +I. We have developed a luminescent probe for Cu+, based on
the binding site of the copper chaperone CusF, a periplasmic protein in Gram negative bacteria. This
protein binds copper with the side chains of two methionines (thioether), one histidine (imidazole) and
one tryptophan (indole). The tryptophan is a very unusual ligand that is bound to copper through a
cation/π interaction.1,2 We have designed a peptide that mimics the binding site of CusF for selective
Cu+ binding and we have grafted on this peptide a DOTA-terbium(III) for light emission. In this
peptide, a tryptophan serves both as a Cu+ ligand and as an antenna for terbium sensitization. This
luminescent probe is able to selectively detect Cu+ among physiological cations.
In this communication, we will present the synthesis, the complexation properties and the
photophysical properties of this probe. We will show how the cation/π interaction modulates the
tryptophan antenna properties and changes the terbium emission.
Figure : Copper(I) binding site of the protein CusF (left) and Tb3+ emission of the probe when Cu+ is
added (right).
1. Xue, Y.; Davis, A. V.; Balakrishnan, G.; Stasser, J. P.; Staehlin, B. M.; Focia, P.; Spiro, T. G.; Penner-Hahn, J. E.;
O’Halloran, T. V. Nat. Chem. Biol. 2008, 4, 107–109.
2. Davis, A. V.; O’Halloran, T. V. Nat. Chem. Biol. 2008, 4, 148–151.
34
Cobalt regulation of the PfTET3 aminopeptidase activity
M. Colomboa, b, c, E. Girard,a, b, c Bruno Franzettia, b, c
a) Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France
b) CEA, DSV, IBS, F-38027 Grenoble, France
c) CNRS, IBS, F-38027 Grenoble, France
TETs are self-compartmentalized dodecameric metalloaminopeptidases belonging to M42 family
devoted to process peptides up to 20 amino acid length. They were first discovered in Archaea1 and
homologs were then identified in Bacteria and Eukaria. To date, three archaeal TETs have been
characterized: TET1 (specific for negatively charged residues), TET2 (cleaves mainly hydrophobic
residues) and TET3 (specific for positively charged residues). Peptide hydrolysis occurred in the
proteolytic domain of each subunit and the reaction is catalysed by two metal ions. It has been shown
that cobalt enhances the activity of numerous peptidases, but its fine activation mechanism is not
completely understood. Here, by studying the hyperthermophilic TET3 from Pyrococcus Furiosus
(PfTET3), insights into PfTET3 enzymatic activity and metal dependency were provided by means of
anomalous X-ray crystallography and UV-spectrophotometry using Lys-pNA as substrate. The effects
of Zn2+ and Co2+ concentration on the enzymatic activity were analysed at T= 20°C, 50°C and 85°C.
Results show that Zn2+ strongly inhibits PfTET3 activity at all T, while Co2+ enhances two times
PfTET3 activity at 85°C and it is inhibitory at lower temperatures. Furthermore, PfTET3 crystals were
grown at T=20°C in the presence/absence of stochiometric amounts of Co2+ and X-ray data were
collected at the absorption edges of Zn2+ and Co2+. Anomalous maps revealed that cobalt replaced one
zinc in the active site that is coordinated by residues His314, Asp177 and Glu208. Moreover, based on
X-ray anomalous map, a third metal site, occupied by Co2+, close to the active site, was detected in the
Co2+ containing crystals. The third site occupies the specificity pocket of the substrate recognition.
These data suggest a specific regulatory role of Co2+ in the substrate turnover of the hyperthermophilic
PfTET3.
1.
B. Franzetti, G. Schoehn, EMBO J. 2002, 21, 2132-2138.
35
Structural and functional analysis of the Ferric Uptake Regulator
(FUR) proteins from various pathogens
E. de Rosnya, J. Perardb, M. Savarda, M. Ould Abeihb, M. Castellana, C. Solarda, S. Mathieub R. Mirasb,
S. Crouzyb, J. Covès,a I. Michaud-Soretb
a) Heavy Metal and Signaling, Metallo group, Institut de Biologie Structurale, UMR 5075 CNRS/CEA/UJF, 6 rue Jules
Horowitz, F-38027 GRENOBLE Cedex 1.
b) BioMet Group and 2MCT Group from the Laboratoire de Chimie et Biologie des Métaux UMR 5249 CNRS/CEA/UJF,
iRTSV-CEA, Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex9
The Ferric Uptake Regulator (Fur) is involved in iron metabolism, including synthesis, recognition and
transport of siderophores but also in the regulation of hundreds of other genes including genes of
virulence factors. When bound to ferrous iron, FUR becomes able to bind to specific DNA sequences,
called FUR boxes. This binding triggers the repression or the activation of gene expression, depending
on the regulated genes. FUR is ubiquitous in proteobacteria and several crystallographic structures of
metal bound FUR have been determined so far (1). Despite high homology in the overall folding,
important variations arise especially in the coordination shell of the metal and in the orientation of the
DNA binding domains. It is therefore of interest to explore FUR from different species in order to
understand the relation between structure and function.
In this study, we have cloned the fur genes from four pathogenic strains Pseudomonas aeruginosa, Legionella
pneumophila, Yersinia pestis, Francisella tularensis which protein sequence homologies range from 38%
(FtFur vs YpFur) to 60% (LpFur vs PaFur). The corresponding proteins were purified and compared
to FUR from E. coli. We found that PaFur, FtFur and LpFur are tetramers in solution; which is in
contradiction with the dogmatic view of FUR being dimers. To study the regulation mechanism of Fur
in relation with its oligomeric state and the open/closed conformations of the DNA binding domains,
multidisciplinary approaches were carried out combining molecular biology, biochemistry and
biophysical studies (size exclusion chromatography, SAXS, SEC-MALLS-RI and cross-linking). We
have showed that the tetrameric forms of those proteins switch from tetramers to dimers upon DNA
binding after metal activation. These data suggest that the dimer is the common active form for all
these proteins. In addition to this structural analysis we have investigated the efficiency of different
metal ions to activate the FUR proteins and found different behaviors, confirming that behind an
apparent similarity the FUR proteins retain some specificity. These differences of efficiency do not
directly correlate with the affinity of the FUR proteins for the metals, determined by competition with
chromogenic chelators. A better understanding of the molecular mechanisms by which “pathogenic”
bacteria acquire iron is significant from public-health perspective because it can lead to the discovery of
new anti-virulent compounds.
1.
MF. Fillat, Arch Biochem Biophys 2014, 546, 41
36
POSTERS
37
38
Liste des posters
1 Béatrice Blanc
Molecular investigation of iron-sulphur cluster assembly scaffolds
under stress
2 Bastien Boff
Bio-inspired tripodal ligands of copper(I)
3 Alexandra Botz
DNA recognition by lanthanide-binding hexapeptides
4 Martine Cuillel
Drug candidates for the Wilson’s disease: a follow-up
5 Pascale Delangle
Targeting of glycoconjugates to hepatic cells for copper chelating
therapy related to Wilson’s disease
6 Eric Gouré
Synthesis and characterization of MnII-CaII complexes
7 Lauréline Lecarme
Interaction of Ni(II)-salen with G-quadruplex DNA
8 Sylvia Lehmann
Evaluation of age impact on the protective effect of selenium
against UVA irradiation in primary human keratinocytes, a
proteomic analysis
9 Caroline Marchi-Delapierre
An Fe based artificial oxygenase
10 Isabelle Michaud-Soret
Interference between NP-TiO2 nanoparticles
and iron homeostasis in E. coli
11 Roger Miras
Patrice Catty
Intrinsic fluorescence study of metal binding to CadA, the Cd2+Zn2+- Pb2+-ATPase of Listeria monocytogenes
12 Mohamed Ould Abeih
Structure-function relationships of FURs and their inhibitors:
towards new antibacterial compounds
13 Adrien Pagnier
Structural and functional studies of an unusual L-cysteine
desulfurase from Archaeoglobus fulgidus
14 Laurent Raibaut
Tidbits for the preparation of polyaminocarboxylate chelates used
to design LnIII based near-infrared luminescent zinc finger probes
15 Deborah Reichmann
New insights into the reaction mechanism of the Fe4S4
Quinolinate Synthase NadA
16 Angélique Troussier
Binuclear hydrolases: Desperate search for a nucleophile
17 Xavier Vernède
AnoXtal: Automated Crystallization of Proteins under Anoxic
Conditions
39
40
Molecular Investigation of Iron-sulphur cluster Assembly Scaffolds
under Stress
B. Blanca,b,c, Martin Clemanceya,b,c, Jean-Marc Latoura,b,c, M. Fontecavea,b,c,d, S. Ollagnier de Choudensa,b,c
a) Université Joseph Fourier, Laboratoire de Chimie et Biologie des Métaux, 17 Rue des Martyrs, 38054, Grenoble, Cedex 09,
France
b) CNRS, Laboratoire de Chimie et Biologie des Métaux, 17 Rue des Martyrs, 38054, Grenoble, Cedex 09, France
c) CEA-Grenoble, iRTSV/CBM, 17 Rue des Martyrs, 38054, Grenoble, Cedex 09, France
d) Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Université Pierre et Marie Curie-Paris 6, Collège de
France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France
Iron-sulphur (Fe-S) clusters are essentials inorganic cofactors that are involved in most cellular
processes. Fe-S biosynthesis is known to be controlled by three machineries in bacteria (NIF, ISC and
SUF). E. coli carries the two ISC and SUF systems and offer the opportunity to study and compare
both systems. Both systems contain similar key molecular actors and share the same basic mechanism
for Fe-S clusters assembly: during the assembly step the Fe-S cluster is formed on a scaffold protein
(SufBC2D and IscU for SUF and ISC machinery respectively) and the transfer step corresponds to Fe-S
transfer from the scaffold to apo-target proteins (Scheme 1). Based on in vivo data, the SUF machinery
is proposed to be involved in Fe-S biosynthesis under oxidative stress whereas ISC functions under
normal conditions. However, so far no molecular data have been obtained strengthening this idea.
What are the intrinsic properties of the SUF system allowing it to be better adapted to build Fe-S
clusters than ISC system under stress conditions? A first possibility is that the key actor in the SUF
machinery, scaffold protein, contains a more stable cluster; a second one is that Fe/S transfer is more
efficient under stress using SUF machinery. The present work focuses on molecular studies of key
actors in Fe-S assembly, the SufB and IscU scaffold proteins under oxidative stress and iron limitation.
We showed that IscU Fe2S2 cluster is less stable than the SufB Fe2S2 cluster in the presence of H2O2
and oxygen. Moreover, SufB cluster is less affected than IscU one to iron chelator likely reflecting less
solvent-exposure. Finally, SufB cluster stability is further enhanced in the presence of its chaperones.
Our results are fully consistent with in vivo observations that SUF system manages to work better under
oxidative conditions than ISC one.
Carriers
Scaffold
Cysteine desulfurase
Apo
Chaperones
IscS, SufSE
S
S
H
IscU, SufBC2D
Cys
Ala
Fe2+
HscA,HscB
SufC, SufD
Holo
e-
Fe-S Assembly
Fe-S Transfer
Scheme 1: Fe-S biogenesis
41
Bio-inspired tripodal ligands of copper(I)
B. Boff, A.-S. Jullien, A. Pujol, C. Lebrun, C. Gateau, P. Delangle
Laboratoire de Chimie Inorganique et Biologique (UMR_E 3 CEA UJF), Institut Nanosciences et Cryogénie, CEA Grenoble,
17 rue des martyrs, 38 054 Grenoble, France.
Metal overload plays an important role in several diseases or intoxications, like in Wilson’s disease, a
major genetic disorder of copper metabolism in humans. Indeed free Cu can promote Fenton-like
reactions and be very toxic even at low concentration. Therefore, intracellular Cu concentration needs
to be rigorously controlled so that it is only provided to the essential enzymes but does not accumulate
to toxic levels.1, 2 In Wilson’s disease, impairment of the copper transport in hepatocytes, results in
cytosolic Cu accumulation with associated cellular injury.3 To efficiently and selectively decrease the
copper concentration in the liver, an organ highly damaged in this disease, chelators should be targeted
at hepatocytes. Since excess copper in hepatocytes is expected to be Cu(I), we are designing selective
and efficient Cu(I) ligands, which are targeted to the liver.4
In this communication, we will present the design of tripodal Cu(I) chelating agents inspired from
proteins involved in Cu homeostasis. They are based on a nitrilotriacetic acid scaffold extended with
three coordinating amino acids. Cysteine derivatives demonstrated a very high affinity for the soft Cu(I)
cation, with the formation of trigonal planar sulfur-only coordinating sites either in a mononuclear
complex or in a cluster with a Cu6S9 core. These thiolate-based ligands share many similarities with
cysteine-rich metallothioneins (MTs). In particular, they reproduce the very high stabilities of Cu(I)MTs complexes with dissociation constants of 10-19 M.5, 6 These tripodal architectures are currently
functionalized with other amino acids found in Cu transporter proteins to evidence the impact of the
coordinating sites onto the nature of the Cu(I) complexes formed in water and their stabilities.
SH
EtOOC
COOH
N
HOOC
H
STrt
COOH + 3
H2N
N
O
H
N
N
COOEt
O
HS
O
COOEt
Cu(I)
O
O
N
EtOOC
NH
EtOOC
NH
O
Cu(I)
HN
COOEt
SH
N
H
COOEt
CuS3
1.
N. J. Robinson and D. R. Winge, Annu. Rev. Biochem. 2010, 79, 537-562.
2.
B. E. Kim, T. Nevitt and D. J. Thiele, Nat. Chem. Biol. 2008, 4, 176-185.
3.
P. Delangle and E. Mintz, Dalton Trans. 2012, 41, 6359-6370.
4.
A. M. Pujol, M. Cuillel, A.-S. Jullien, C. Lebrun, D. Cassio, E. Mintz, C. Gateau and P. Delangle,
Angew. Chem. Int. Ed. 2012, 51, 7445-7448.
5.
A. M. Pujol, C. Gateau, C. Lebrun and P. Delangle, J. Am. Chem. Soc. 2009, 131, 6928-6929.
6.
A. M. Pujol, C. Gateau, C. Lebrun and P. Delangle, Chem. Eur. J. 2011, 17, 4418-4428.
42
Cu6S9
DNA recognition by lanthanide-binding hexapeptides
A. Botz, L. Ancel, C. Lebrun, C. Gateau, P. Delangle
Univ. Grenoble Alpes, INAC, SCIB, RICC F-38000 Grenoble, France
CEA, INAC, SCIB, Laboratoire de Reconnaissance Ionique et Chimie de Coordination F-38054 Grenoble, France.
Luminescence is a very efficient technique to detect supramolecular interactions between molecules and
DNA. It has to be enough sensitive to overcome the natural fluorescence of biological material.
Lanthanide ions, with their unique spectroscopic properties like their long-lived excited state and
narrow band emission, prove to be powerful tools to detect DNA.
In this context, we are designing efficient lanthanide-peptides complexes to detect the interaction with
DNA through the metal-centered luminescence. The design of such probes required the development
of ligands which can effectively coordinate the metal ions, sensitize these metal ions by antenna effect
and induce a change in the luminescence of the lanthanide in the presence of DNA.
It has been demonstrated that hexapeptides containing two natural amino acids bearing aminodiacetate
side chains and a sequence Pro-Gly provide LnIII-peptide complexes with high-enough stability to avoid
dissociation in water at physiological pH. In fact, the Pro-Gly sequence promotes a β-turn
conformation of the peptide, enhancing the coordination of the cation thanks to a spatial arrangement
between the two chelating groups of the unnatural amino acids.
Furthermore, organic fluorophores coupled to the N-terminus of these peptides act as intercalating
agents and sensitizer of the lanthanide cations. Upon excitation of the sensitizer unit without DNA, the
energy transfer from this unit to the lanthanide ion by antenna effect highlights the specific
luminescence of the metal. In the presence of DNA, the sensitizer unit intercalates into this
biomolecule and the energy transfer is no more possible, which significantly affects the metal-centered
luminescence (cf. figure 1).
Figure 1 : detection of Ct-DNA by a lanthanide-binding peptide
1. F. Cisnetti, C. Gateau, C. Lebrun, P. Delangle, Chem. Eur. J. 2009, 15, 7456-7469
2. F. Cisnetti, C. Lebrun, P. Delangle, Dalton trans. 2010, 39, 3560-3562
3. A. Niedzwieck, F. Cisnetti, C. Lebrun, P. Delangle, Inorg. Chem. 2012, 51, 5458-5464
4. A. Niedzwiecka, F. Cisnetti, C. Lebrun, C. Gateau, P. Delangle, Dalton Trans. 2012, 41, 3239-3247
5. L. Ancel, C. Gateau, C. Lebrun, P. Delangle, Inorg. Chem. 2013, 52, 552-554
43
Drug Candidates for the Wilson’s Disease: a follow-up
M. Cuillela, P. Charbonniera, A. Harela, E. Mintz,a V. Brunb, M. Jaquinodb
P. Delanglec, A. Pujolc, A.S. Jullienc, C. Gateauc, C. Lebrunc, C. Rivauxc
a) Laboratoire Chimie et Biologie des Métaux (UMR 5249 CEA/CNRS/Univ Grenoble Alpes) - iRTSV- CEA
Grenoble.
b) Laboratoire Biologie à Grande Echelle (U 1038 CEA/Univ grenoble Alpes) - iRTSV- CEA Grenoble
c) Laboratoire Chimie Inorganique et Biologique (UMR E3 CEA/ Univ Grenoble Alpes) –INAC - CEA Grenoble
The Wilson’s disease is a major disorder of copper metabolism in humans due to mutations on the
ATP7B gene. Impairment of the Cu transport in hepatocytes results in cytosolic Cu accumulation with
cellular injury. Current drugs are not always satisfying and hepatic transplant may be necessary. Since
the pool of intracellular copper is in the +I oxidation state, we figured that specific chelators for Cu(I)
that would enter the hepatic cells could potentially represent an improving alternative.
Therefore, we designed bifunctional molecules able to both efficiently complex Cu(I) and target
hepatocytes: Chel1 and Chel2 .
Various studies are reported, in cellulo1-2 and in vivo, to check whether the chelators are reliable drug
candidates.
1.
Pujol, Cuillel, Renaudet, Lebrun, Charbonnier, Cassio, Gateau, Dumy, Mintz, Delangle. J Am Chem Soc (2011) 133,
286
2.
Pujol, Cuillel, Jullien, Lebrun, Cassio, Mintz, Gateau, Delangle. Angew Chem (2012) 51, 7445.
44
Targeting of glycoconjugates to hepatic cells for copper chelating
therapy related to Wilson’s disease
P. Delangle,a M. Monestiera,b, C. Rivauxa, C. Lebrun, C. Gateaua, O. Renaudetb, P. Charbonnierc, M.
Cuillelc, E. Mintzc.
a) CEA, Univ. Grenoble Alpes, INAC, SCIB, RICC, F-38054 Grenoble, France
b) CNRS, Univ. Grenoble Alpes, DCM, I2BM, F-38041 Grenoble, France
c) CEA, CNRS, Univ. Grenoble Alpes, IRTSV, LCBM, Biomet, F-38054 Grenoble, France
Copper homeostasis dysfunction is responsible for the Wilson’s disease. In this genetic disorder there is
a Cu overload in the liver, which is the key organ for Cu delivery and excretion in the body. This
accumulation leads to the destruction of the tissues by oxidative stress. Current therapies mainly aim at
limiting copper absorption and enhancing its excretion but there are many side effects due to their lack
of specificity. In this context, we propose an innovative strategy that would selectively detoxify copper
in liver cells. Since excess intracellular Cu is in the +I oxidation state, we figured that a chelator that
would enter the hepatic cells and be specific for Cu(I) could represent an efficient strategy. Two
glycoconjugates Chel1 and Chel2 that contain a Cu(I) chelating unit associated to liver targeting units
have been obtained and demonstrated to enter hepatocytes and chelate intracellular copper.[1]
The chelating units are cysteine-rich compounds and can be considered as prodrugs since their
coordinating thiol groups are hidden in disulfide bridges, which are reduced only after entering the
targeted cells. The targeting units are directed to an abundantly and exclusively expressed lectin located
at the surface of the hepatocyte’s membrane: the asialoglycoprotein receptor (ASPG-R). The
recognition of Chel1 and Chel2 with the ASGP-R is allowed by their functionalization with several Nacetyl-galactosamine (GalNAc) residues.[2-4] The efficiency of the targeting systems of Chel1 and
Chel2 will be compared in this communication.
1.
2.
3.
4.
P. Delangle and E. Mintz, Dalton Trans., 2012, 41, 6359-6370.
A. M. Pujol, M. Cuillel, A.-S. Jullien, C. Lebrun, D. Cassio, E. Mintz, C. Gateau and P. Delangle, Angew. Chem. Int. Ed.,
2012, 51, 7445-7448.
A. M. Pujol, M. Cuillel, O. Renaudet, C. Lebrun, P. Charbonnier, D. Cassio, C. Gateau, P. Dumy, E. Mintz and P.
Delangle, J. Am. Chem. Soc., 2011, 133, 286-296.
C. Gateau and P. Delangle, Ann. N.Y. Acad. Sci., 2014, doi: 10.1111/nyas.12379.
45
Synthesis and characterization of MnII-CaII complexes
B. Gerey, M. Gennari, E. Gouré, J. Pécaut, F. Molton, C. Duboc, J. Fortage, M.-N. Collomb
DCM – équipe CIRE, UFR de chimie Bat. C BP53 38041 GRENOBLE cedex 9
The goals of this project are to improve the current knowledge on the structure and function of the
OEC by developing a new family of homo- and hetero-nuclear complexes of Manganese and Calcium
incorporating aquo/hydroxo terminal ligands and carboxylates ligands. To access these complexes, our
strategy is to develop a new family of multidentate ligands incorporating the pyridine-carboxylate motif.
Carboxylate groups will not only better mimic the coordination sphere of the OEC, but also promote
the complexation of alkali-earth ions such as calcium. The aim is also to study their electrochemical and
spectroscopic properties as well as their reactivity.
46
Interaction of Ni(II)-salen with G-quadruplex DNA
L. Lecarme a, E. Prado a, A. De Rache b, M.L. Nicolau-Travers c, H. Jamet a, A. Van Der Heyden a, E.
Defrancq a, D. Gomez c, J.L. Mergny b, O. Jarjayes a, F. Thomas a
a) University of Grenoble, Department of Molecular Chemistry, F-38041 Grenoble Cedex 9 (France)
b) Univeristy of Bordeaux, European Institute of Chemistry and Biology, F-33607 Pessac (France)
c) CNRS, Institute of Pharmacology and Structural Biology, F-31077 Toulouse Cedex 4 (France)
Guanine-rich sequences of DNA can self-assemble into specific tetrastranded structures (Gquadruplexes), which feature stacked tetrads of guanines.1 G-rich regions have been identified in the
telomeres (extremity) of chromosomes and are believed to be essential for chromosomal maintenance
by Telomerase.2 This enzyme is over-expressed in most of the cancer cells. It protects chromosomes
against a natural trimming resulting in replication and provides immortality to tumour cells. If DNA is
folded into G-quadruplex structures, Telomerase is inhibited.
G-quadruplexes are also found in promoter regions of some oncogenes, suggesting that they are
also involved in gene regulation and the appearance of certain cancers. The development of new drugs
which target specifically and stabilize G-quadruplex structures is a field of growing interest.3
Compounds having these properties would act as Telomerase inhibitors, and consequently constitute
potential anti-cancer drugs. In addition they may be important tools to understand the folding and
identify G-quadruplexes in vivo.
Metal complexes and especially cationic square planar salophen complexes (see figure) provide a
versatile platform to develop G-quadruplex intercalators.4
We describe in this communication the rational design (by docking) of salen ligands which
exhibit a high affinity for G-quadruplexes, as well as their synthesis. New results of Surface Plasmon
Resonance evidence a high selectivity towards quadruplex DNA over duplex, and results of Telomeric
Repeat Amplification Protocol shows an inhibition of telomerase activity.
1. G. N. Parkinson, M. P. H. Lee, S. Neidle, Nature 2002, 417, 876.
2. E. H. Blackburn, Angew. Chem. Int. Ed. 2010, 49, 2.
3. S. N. Georgiades, N. H. Abd Karim, K. Suntharalingam, R. Vilar, Angew. Chem. Int.Ed. 2010, 49, 4020.
4. J.E. Reed, A. Arola-Arnal, S. Neidle, R. Vilar, J. Am. Chem. Soc. 2006, 128, 5992.
47
Evaluation of age impact on the protective effect of selenium against
UVA irradiation in primary human keratinocytes, a proteomic analysis
W. Rachidiab, C. Favrotab, S. G. Lehmannacd, M. Seveacd
a) Université Grenoble Alpes (UGA)
b) Laboratoire Lésions des Acides Nucléiques, SCIB,CEA, Grenoble
c) INSERM, IAB, Plateforme Protéomique PROMETHEE, Grenoble, France
d) CHU de Grenoble, IAB, Institut de Biologie et de Pathologie, Plateforme Protéomique PROMETHEE, Grenoble, France
Few studies have focused on the protective role of selenium (Se) on skin aging and photoaging
although selenoproteins are essential for keratinocyte function and skin development.
To our knowledge, the impact of Se supplementation on skin cells obtained from elderly and
young donors has not been reported. So, the main objective of our work was to evaluate the effects of
Se supplementation (as its inorganic form sodium selenite (NaSe)) on skin keratinocytes at baseline or
after exposure to UVA irradiation and to identify the impact of such treatment on the proteome.
Keratinocytes were obtained from normal skin biopsies of elderly (60-70 years old) or young
(20-30 years old) donors and were pre-treated with 30 or 240 nM of NaSe for 72h, followed or not by
different doses of UVA. Se concentrations were determined by ICP-MS, cell survival was examined by
using both MTT and clonogenic cell survival assay, activities of cytosolic GPx (GPx1) and cytosolic
TrxR were determined spectrophotometrically and DNA repair capacities were evaluated by a
multiplexed DNA biochip patented and developed at our laboratory. An iTRAQ quantitative
proteomics approach was applied to determine the proteins, which were over- or under-expressed
depending on treatments applied.
We showed that low doses of NaSe (30 nM) were very potent protector against UVA-induced
cytotoxicity on young keratinocytes, whereas the protection efficiency of NaSe on old keratinocytes was
obtained only at higher concentrations (240 nM). Also, we have shown a drastic fall in DNA repair
capacities on old keratinocytes versus young one at basal state or after UVA exposure. Supplementation
with selenium enhances significantly global DNA repair capacities, especially on those isolated from
young donors. It seems that selenium is able to increase OGG1 activity, a glycosylase responsible for
the repairing of 8-oxoGua. The quantitative proteomic approach identified up to 56 proteins whose
expression was modified in different conditions of treatment. We performed an ontology enrichment
and pathway analysis and the top functions identified in elderly vs old were related to dermatological
disease and in selenium and UV treatment in young to wound healing pathways.
These original data strongly suggest an increased vulnerability of keratinocytes with age against
photoaging and should be taken into account regarding Se needs in elderly. Strengthening of DNA
repair activities by selenium may represent a new strategy to fight against aging and skin photo-aging.
These results will highlight the protective mechanism of selenium, and therefore, could identify new
targets for UVA exposure protection.
This work was supported by a grant from Labcatal Company, Montrouge, France.
48
An Fe based artificial oxygenase
Caroline Marchi-Delapierrea, Laurianne Rondota, Adeline Jorge Robina, Christine Cavazzaa, and
Stéphane Ménagea.
a) Laboratoire de Chimie et Biologie des métaux. UMR 5249 Université Joseph Fourier, CNRS et CEA – CEA-Grenoble 17, Avenue des Martyrs, 38054 Grenoble
One of the research activities of the BioCE team concerns the development of catalytic oxidation
reactions that mimic the activity of oxygenases in order to perform oxidation reactions following the
green chemistry principles. The team then invested in the design of new bio-inorganic catalysts:
artificial metalloenzymes. This allows us to find an alternative to polluting solvents (such as water),
toxic and hazardous chemicals.
In this context, we have developed two new biocatalysts with NikA as the protein scaffold. One, an
artificial oxydase, is capable of epoxidation catalysis, in organic media, in the presence of iodobenzene
diacetate. The other, an artificial oxygenase, incorporates a Fe (III) complex is able to activate
molecular oxygen which is the cleanest oxidant and to transfer it to a substrate of interest.
We so have transposed the oxidation reactivity to the corresponding hybrids, affording it milder and
greener condition. Our catalysis results are supported by X-rays analysis on protein crystals.
I will present you the design of the corresponding hybrids and our latest results concerning their in
alkene oxidation catalysis.
49
Interference between NP-TiO2 nanoparticles
and iron homeostasis in E. coli
C. Fauquant1, C. Sageot1, A. Chan1, M. Chevallet1, A.-N. Petit1, N. Herlin-Boime2, P.-H. Jouneau3,
S. Ollagnier de Choudens,1 I. Michaud-Soret1,
(1) Laboratoire de Chimie et Biologie des Métaux UMR 5249 CEA-CNRS-UJF, 38054 Grenoble Cedex 9
(2) Laboratoire Edifices Nanométriques URA 2453 CEA-CNRS-IRAMIS, CEA Saclay 91191 Gif sur Yvette
(3) Laboratoire d'Etude des Matériaux par Microscopie Avancée, Minatec Campus, UMR-E, CEA INAC/UJF-Grenoble1,
Grenoble, 38054, France
e-mail [email protected]
TiO2 nanoparticles (TiO2-NP) production has massively increased during the last decade as well as
their use in commercial products. As a consequence, a major concern has been to address the potential
toxicity of the TiO2-NP on human health as well as on the environment. Studies using uncoated wellcharacterized TiO2-NP have shown that these NP were cytotoxic, and induce an increase of the level of
reactive oxygen species (ROS) in E. coli. (1)
The purpose of our work was to investigate the molecular mechanisms leading to cellular effects of
TiO2-NP especially in relation with the potential disruption of iron homeostasis using E. coli as
prokaryote model since oxidative stress and iron homeostasis are linked.
We firstly defined procedures to disperse the TiO2-NP as well as to determine the exposure
conditions in which our nanoparticles were well-dispersed in a specific cellular growth medium where a
bacterial growth is still observed and not only a survival. (2)
Secondly we studied in these define conditions the impact of a nanoparticle exposure on the bacterial
growth and viability of several strains and mutants (in order to exacerbate phenotypes) on genes related
to iron homeostasis and oxidative stress.
We also evaluated the localization of nanoparticles in presence of bacteria and quantify iron and
titanium content.
In addition, we studied the chelation of Ti(IV) by enterobactin siderophore and its solubilization
from TiO2-NP by action of enterobactin.
Finally a targeted study was performed to analyze the impact of nanoparticle exposure on enzymatic
activities measured in cellular extracts. These targeted enzymes have metallic sites (iron-sulfur cluster)
and can be potential biomarkers of a disturbance of the iron homeostasis.
Altogether, we obtained original data that will be presented here suggesting how metallic
nanoparticles such as (TiO2-NP) could interfere with iron homeostasis.
1.
2.
Simon-Deckers, A. et al. Environ. Sci. Technol. 2009, 43, 8423–8429.
Herlin-Boime N, Michaud-Soret I, Fauquant C, Armand L and Carrière M. From the synthesis of TiO2 nanoparticles to
the study of their behavior. Biofutur, 2013, 347: 39-41
50
Intrinsic fluorescence study of metal binding to CadA, the Cd2+-Zn2+Pb2+-ATPase of Listeria monocytogenes
R. Miras, P. Catty
Laboratoire de Chimie et Biologie des Métaux
UMR CEA-UGA-CNRS n°5249
17, rue des Martyrs
38054 GRENOBLE Cedex 9
e-mail : [email protected] [email protected]
P-type ATPases are widespread membrane proteins involved in the transport of numerous
ions (Ca2+, Na+, K+, H+, Cu+, Cu2+, M2+, Zn2+, Pb2+ and Cd2+) and for some of them,
aminophospholipids 1, 2. Found in all types of living organisms and classified according to their
topology or their primary sequence homology, P-type ATPases participate in a large variety of
cellular processes among them metal homeostasis 3.
The Cd2+-ATPase CadA from the intracellular pathogen Listeria monocytogenes, described as an
essential determinant of the resistance to Cd 4, has been extensively studied in our group. We notably
focused our research on two aspects of CadA functioning: (i) the role, the selectivity and the NMR
structure of its N-terminal metal binding domain 5; (ii) the characterization of its Cd2+ transport sites 6.
More recently, we used CadA as a tool to study cadmium toxicity in yeast 7.
To gain further insights into the enzymatic and structural features of CadA, we purified it after
expression in E. coli using the StrepTag. ATPase activity measurements and intrinsic fluorescence
studies revealed distinct behaviours of the transporter in the presence of Zn2+, Pb2+ and Cd2+. In
addition, these measurements were found in agreement with the hypothesis of 2 metal ions
transported per ATP hydrolysis, a hypothesis previously deduced from biochemical experiments on
the non-purified protein expressed in Sf9 cells 6.
1.
2.
3.
4.
5.
6.
7.
KB Axelsen, MG Palmgren, J Mol Evol 1998, 46: 84-101.
W Kuhlbrandt, Nat Rev Mol Cell Biol 2004, 5: 282-295.
JM Arguello, J membr Biol 2003, 195: 93-108.
M Lebrun, A. Audurier, P. Cossart, J Bacteriol 1994 176: 3040-3048.
L Banci et al., J Mol Biol 2006 356: 638-650.
CC Wu et al., J Biol Chem 2006 281: 29533-29541.
A Gardarin et al., Mol Microbiol 2010 76: 1034-1048.
51
Structure-function relationships of FURs and their inhibitors: towards
new antibacterial compounds
M. Ould Abeih1 , S. Mathieu1, C. Cissé1,2, S. Vitale1, J. Perard1, M. Castellan3, L. Flanagan2, S. Galop1,
R. Miras1, C. Solard3, P. Catty1, D. Boturyn4, J. Covès3, E. de Rosny3, S. Crouzy2, I. Michaud-Soret1
1BioMet
and 2MCT Groups from the Laboratoire de Chimie et Biologie des Métaux UMR 5249 CNRS-CEA-UJF, Grenoble,
3IBS
4DCM,
UMR5275, CEA-CNRS-UJF, Grenoble,
301, rue de la chimie, Saint Martin d’Hères, France
e-mail : [email protected]; [email protected]
Overuse of antibiotics induced the development of resistant pathogens which are becoming a real
public health problem. New antibacterial compounds and targets must be found. A new approach to
avoid the rapid emergence of antibiotic resistance is to act on targets impacting virulence. The
regulation of iron uptake systems is in this context, a promising target. Iron is an essential element,
toxic in high doses, whose the concentration must be tightly regulated. The major player in this
regulation is the FUR (Ferric Uptake Regulator), a global transcription regulator, able, once metallated
with iron, to bind DNA regions that control the expression of genes involved in iron homeostasis,
oxidative stress and virulence factors. FUR is ubiquitous in Gram-negative bacteria and absent in
eukaryotes, FUR is an interesting target to fight against bacterial infections since the inactivation of fur
gene in various pathogens leads to virulence decrease.
Peptidic aptamer technology was used to select, in a 20 million-molecule library, 4 inhibitors interacting
with E. coli FUR (EcFur). Each molecule consists of a variable 13 amino acid peptide loop attached at
both ends to a protein scaffold. These molecules were able to decrease pathogenic E. coli strain
virulence in a fly infection model. To characterize the interaction between the variable peptidic loops
and EcFur protein, more than 20 peptide derivatives have been synthesized. In vitro activity assays
were performed to determine IC50, to classify peptides with respect to each other and to identify
minimal active sequences and essential residues. Submicromolar dissociation constants of
EcFur/peptide interaction were determined by isothermal microcalorimetry. Molecular modelling and
docking experiments of various peptides on homology models of EcFur were performed to suggest an
inhibition pocket in Fur as peptide binding site. The location of this pocket was checked by both two
hybrid and activity assays. Finally, these peptides were also tested against other Fur proteins from
different pathogens to identify broad spectrum inhibitory molecules.
52
Structural and functional studies of an unusual L-cysteine desulfurase
from Archaeoglobus fulgidus
A. Pagnier, L. Zeppieri, Y. Nicolet, J.C. Fontecilla-Camps
Institut de Biologie Structurale, 6 rue Jules Horowitz, 38000 Grenoble
L-Cysteine desulfurase IscS and scaffold IscU proteins are universally involved in Fe/S cluster
synthesis. The Archaeoglobus fulgidus (Af) genome encodes proteins having a high degree of primary
structure similarity to IscS and IscU from other organisms. However, AfIscS is unusual because it lacks
the active site lysine residue that normally forms an internal Schiff base with pyridoxal-phosphate (PLP)
and serves as a base during catalysis. This residue is replaced by Asp199 in AfIscS. The as-isolated
native recombinant AfIscS lacks desulfurase activity and contains pyridoxamine phosphate (PMP),
instead of the expected PLP, which binds non-covalently at the PLP site of the enzyme as shown by its
crystallographic structure solved to 1.43 Å resolution 1.
Here, we report the 1.8 Å resolution structure of the K199D AfIscS variant does not form the expected
Schiff base either. We also provide functional data that shows AfIscS is essential for cluster assembly
not as a sulfide provider but because, as shown by the X-ray structure of the Af (IscU-D35A-IscS)2
complex structure 2, its active site cysteine residue is a ligand of the nascent Fe/S cluster.
1.
Y. Yamanaka, et al., Dalton Transactions 2012, 42, 3092-3099.
2.
E. Marinoni, et al., Angew Chem Int Engl 2012, 51, 5439-42.
53
Tidbits for the preparation of polyaminocarboxylate chelates used to
design LnIII based near-infrared luminescent zinc finger probes
L. Raibauta, M. Isaaca, C. Lebrunb, J.-M. Latoura, O. Sénèquea
a) LCBM/PMB, Univ. Grenoble Alpes/CNRS/CEA, 38054 Grenoble
b) iNAC/SCIB/RICC, CEA, 38054 Grenoble
Metal ions are essential for life. In particular Zn2+ is widely required in cellular functions, and its
dysregulation is implicated in neurodegenerative diseases or cancer. A major comprehension of the
physiological role of zinc required new tools to image zinc and its flux in living organisms. Interestingly,
a class of proteins named zinc fingers binds specifically Zn2+ in which the metal plays a structural role
contributing to the stability of the domain. Advantageously, we can exploited this property to design
new smart zinc probes operating on the basis of Ln3+ ions emitting in the near-infrared and of zinc
finger peptides for the selective binding of Zn2+ to ensure specific and sensitive response of the probe.
The design of these luminescent probes involves the grafting of Ln3+ complex on a zinc finger
peptide. In this communication, we will report on the efficient synthesis of macrocyclic derivates based
on cyclen, cyclam or triethylene (tri or tetra) amine. Preliminary models of zinc probes were developed
using DOTA or DTPA as ligand for Eu3+ or Tb3+ lanthanides. Nevertheless, in the case of nearinfrared lanthanide (Yb3+, Nd3+) their luminescence can be quenched by the surrounding water
molecules in the coordination sphere and so required other ligands (TETA, DO3A-pic, TTHA) giving
an efficient shielding of the metal ions.
54
New insights into the reaction mechanism of the Fe4S4 Quinolinate
Synthase NadA
D. Reichmanna, A. Chana, M. Cherrierb, P. Amarac, J. Fontecilla-Campsc, S. Ollagnier de Choudensa
a) iRTSV/CBM, 17 rue des martyrs, 38045 Grenoble.
b) University Lyon 1/IBCP, 7 passage du Vercors, 69367 Lyon
c) IBS/Metalloproteins, 6 rue Jules Horowitz, 38000 Grenoble
Nicotinamide adenine dinucleotide (NAD) is an essential cofactor playing a crucial role in several
biological redox reactions1. In the NAD biosynthesis a common precursor exists among all organisms,
the quinolinic acid (QA), which in most prokaryotes is generated from L-aspartate and
dihydroxyacetone phosphate (DHAP). In the well characterized first step L-aspartate is oxidized by the
L-aspartate oxidase NadB to iminoaspartate (IA). In contrast to this the following condensation of IA
with DHAP, carried out by the quinolinate synthase NadA, is still poorly understood at the molecular
level. The NadA protein contains an oxygen sensitive Fe4S4 cluster which is essential for activity2.
Interestingly the cluster is coordinated only by three cysteines, leading to the supposition that the
fourth iron site is involved in the reaction mechanism3. This hypothesis was recently supported by the
use of an inhibitor which binds the cluster4.
Here we present the first available crystal structure of holo-NadA in the presence of its Fe4S4 cluster5.
Based on the structure and a sequence alignment, a variety of strictly conserved amino acids were
changed and the corresponding variants characterized with the aim to give new insights into the
reaction mechanism of NadA.
1.
T.P. Begley, C. Kinsland, R.A. Mehl, A. Ostermann, P. Dorrestein, Vitam. Horm. 2001, 61, 103.
2.
S. Ollagnier de Choudens, L. Loiseau, Y. Sanakis, F. Barras, M. Fontecave, FEBS Lett. 2005, 579, 3737.
3.
C. Rousset, M. Fontecave, S. Ollagnier de Choudens, FEBS Lett. 2008, 582, 2937.
4.
A. Chan, M. Clémancey, J.M. Mouesca, P. Amara, O. Hamelin, J.M. Latour, S. Ollagnier de Choudens Ang. Chem. 2012,
51, 7711.
5.
M.V. Cherrier, A. Chan, C. Darnault, D. Reichmann, P. Amara, S. Ollagnier de Choudens, J.C. Fontecilla-Camps, JACS
2014, Epub ahead of print.
55
Binuclear hydrolases: Desperate search for a nucleophile
E. Gouréa, M. Carbonia, A. Troussierb, C. Lebrunc,d, P. Dubourdeauxa, M. Clémanceyb, G. Blondine,
J.-M. Latoura
a) CEA, DSV, IRTSV, LCBM, PMB, F-38054 Grenoble, France
b) University of Grenoble Alpes, LCBM, F-38054 Grenoble, France
c) CEA, DSM, INAC, SCIB, RICC, F-38054 Grenoble, France
d) University of Grenoble Alpes, LCIB, UMR-E3, F-38054 Grenoble, France
e) CNRS UMR 5249, LCBM, F-38054 Grenoble, France
Many biological hydrolytic reactions are performed by binuclear metal hydrolases with a wide
variety of metal ions: Mn, Fe, Ni, Zn. The most widely studied of these enzymes are the Purple Acid
Phosphatases which hydrolysize mono-or diphosphate esters.[1] Depending on the different isozymes,
their active sites consist of a pair FeIIIMII with MII = FeII, MnII, ZnII.[2] In spite of a huge number of
experimental and theoretical studies of the enzymes and model complexes, the nature of the active
nucleophile involved in these hydrolytic reactions is still a matter of intense debate.
To address this question, we have performed spectroscopic and reactivity experiments using the
following binuclear FeIII FeII complex.
1.0
1.0
A
B1
B2
C
v0
8
8
0.8
5
6
7
8
9
10
pH
Figure 1: Structure of Fe Fe complex
4
0.4
–1
0.0
II
0.6
8
4
0.4
0.2
III
6
2
2
0
0
Relative abundance
6
0.6
10 .v0 (M.s )
Relative abundance
0.8
0.2
0.0
5
6
7
pH
Figure 2: pH dependent speciation and activity
The very rich spectroscopic properties of these FeIII FeII complexes enable us to use for the first
time 1H-NMR and Mössbauer to determine the species present in solution over the pH range 5 to 10.
Comparison of the speciation curves with the reactivity profile reveals a major role of the bridging
hydroxide in the hydrolysis process.
1.
2.
Schenk, G. ; Mittic, N. ; Hanson, G. R.; Comba, P. Coord. Chem. Rev. 2013, 257, 473
Gahan, L. R.; Smith, S. J.; Neves, A.; Schenk, G. Eur. J. Inorg. Chem. 2009, 2745
56
AnoXtal: Automated Crystallization of Proteins under Anoxic
Conditions
A.-M. Villard, C. Darnault, L. Martin, X. Vernède, J. C. Fontecilla-Camps
Groupe Métalloprotéines, Institut de Biologie structurale, Grenoble, France
http://www.ibs.fr/plates-formes/autres-instruments-et/cristallisation-des-proteines-sous/
e-mail: [email protected]
AnoXtal is a platform dedicated to proteins crystallization under anoxic conditions for their structural
analysis. Automation allows for the high throughput screening of hundreds of crystallization
conditions.
The AnoXtal platform is opened to all users and consists of two main instruments:
• An automated crystallization robot: Gryphon (ArtRobbins Inst.),
o 13 commercial screens of 96 conditions can be tested
o A nano-needle delivers 200, 300 or 400 nL protein drops
o Up to three protein samples per plate
•
A semi-automatic device to visualize crystallization plates: CrysCam (ArtRobbins Inst.)
o The crystallization plates are stored in the glove box at 20.5°C
o Six visualization times: J0, J3, J7, J15, J30, and 3 months after setting up the plates
o Images of the corresponding tested conditions are sent to the users
This system is in a hygrometric (>45%) and temperature-regulated (20.5°C+/-0.2°C) glove box
(Jacomex), under an anoxic atmosphere (<20ppmO2)
As member of the “Partnership for Structural Biology” (PSB), we work in collaboration with the HTX
lab from EMBL-Grenoble, France.
Keywords : Glove box, Anaerobe, Crystallization, Robot, Nanovolume
57
58
COORDONNEES
59
60
Liste des participants
Amara Patricia
IBS / METALLO
[email protected]
Arnaud Josiane
CHU / BHN
[email protected]
Blanc Béatrice
LCBM / BIOCAT
[email protected]
Boff Bastien
SCIB / RICC
[email protected]
Borel Franck
IBS / SYNCROTRON
[email protected]
Botz Alexandra
SCIB / RICC
[email protected]
Carriel Diego
EMBL / UVHCI
[email protected]
Castillo Ester
DCM / CIRE
[email protected]
Catty Patrice
LCBM / BIOMET
[email protected]
Cavazza Christine
LCBM / BIOCAT
[email protected]
Cepeda Céline
DCM / I2BM
[email protected]
Chabert Valentin
LCBM / PMB
[email protected]
Champelovier Pierre
CHU / DACP
[email protected]
Chan Alice
LCBM / BIOCAT
[email protected]
Chavarot-Kerlidou Murielle
LCBM / BIOCAT
[email protected]
Colombo Matteo
IBS / METALLO
[email protected]
Covès Jacques
IBS / METALLO
[email protected]
Crouzy Serge
LCBM / MCT
[email protected]
Cuillel Martine
LCBM / BIOMET
[email protected]
De Rosny Eve
IBS / METALLO
[email protected]
Delangle Pascale
SCIB / RICC
[email protected]
Duboc Carole
DCM / CIRE
[email protected]
Esmieu Charlène
LCBM / BIOCE
[email protected]
Fontecave Marc
LCBM & Collège de France
[email protected]
Fontecilla Juan
IBS / METALLO
[email protected]
Gerey Bertrand
DCM / CIRE
[email protected]
Gerez Catherine
LCBM / BIOCAT
[email protected]
Gouré Eric
DCM / CIRE
[email protected]
Hureau Christelle
LCC Toulouse
[email protected]
Iali Wissam
LCBM / BIOCE
[email protected]
Ianello Marina
LCBM / BIOCAT
[email protected]
Isaac Manon
LCBM / PMB
[email protected]
Jacquet Margot
DCM / CIRE
[email protected]
Jarjayes Olivier
DCM / CIRE
[email protected]
Kaeffer Nicolas
LCBM / BIOCAT
[email protected]
Lebrun Vincent
LCBM / PMB
[email protected]
Lecarme Lauréline
DCM / CIRE
[email protected]
61
Lecomte Nicolas
DCM / CIRE
[email protected]
Lehmann Sylvia
IAB
[email protected]
Marchi-Delapierre Caroline
LCBM / BIOCE
[email protected]
Maury Olivier
ENS Lyon / LC
[email protected]
Menage Stéphane
LCBM / BIOCE
[email protected]
Michaud-Soret Isabelle
LCBM / BIOMET
[email protected]
Miras Roger
LCBM / BIOMET
[email protected]
Moreau yohann
LCBM / MCT
[email protected]
Moutet Jules
DCM / CIRE
[email protected]
Neyrolles Olivier
IBPS Toulouse
[email protected]
Nicolet Yvain
IBS / METALLO
[email protected]
Nitschke Wolfgang
BIP Marseille
[email protected]
Nivière Vincent
LCBM / BIOCAT
[email protected]
Ollagnier Sandrine
LCBM / BIOCAT
[email protected]
Ould Abeih Mohamed
LCBM / BIOMET
[email protected]
Pagnier Adrien
IBS / METALLO
[email protected]
Perard Julien
LCBM / BIOMET
[email protected]
Policar Clotilde
ENS Paris / LB
[email protected]
Pradas Ana
ISTerre
[email protected]
Puch Florence
CHU / BHN
[email protected]
Raibaut Laurent
LCBM / PMB
[email protected]
Rat Stéphanie
LCBM / BIOCAT
[email protected]
Reichmann Debora
LCBM / BIOCAT
[email protected]
Rohac Roman
IBS / METALLO
[email protected]
Rondot Laurianne
LCBM / BIOCE
[email protected]
Rull-Barull Jordi
LCBM / BIOCE
[email protected]
Sarret Géraldine
ISTerre
[email protected]
Sautron Emeline
PCV
[email protected]
Seigneurin-Berny Daphné
PCV
[email protected]
Sénèque Olivier
LCBM / PMB
[email protected]
Serre Doti
DCM / CIRE
[email protected]
Sève Michel
IAB
[email protected]
Thomas Fabrice
DCM / CIRE
[email protected]
Torelli Stéphane
LCBM / BIOCE
[email protected]
Troussier Angélique
LCBM / PMB
[email protected]
Vernède Xavier
IBS / METALLO
[email protected]
Ziani Widade
IBS / METALLO
[email protected]
62
Liste des Laboratoires
LCC
Laboratoire de Chimie de Coordination, UPR 8241 CNRS, 205 route de Narbonne,
31077 Toulouse Cedex 4
IBPS
Institut de Pharmacologie et de Biologie Structurale, CNRS - Université de Toulouse,
Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse Cedex 04
BIP
Equipe Evolution de la Bioénergétique, Laboratoire de Bioénergetique et Ingenierie
des Protéines, UMR 7281, CNRS, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex
20
ENS Lyon / LC
Laboratoire de chimie, UMR5182 CNRS – Université de Lyon 1 - ENS Lyon, 46,
allée d'Italie, 69364 Lyon cedex 07
ENS Paris / LB
Laboratoire des Biomolécules, Ecole Normale Supérieure, PSL Research University,
Département de Chimie, 24 rue Lhomond F-75005 Paris
IBS / METALLO
Groupe Métalloprotéines, Institut de Biologie Structurale J. P. Ebel, UMR 5075
CEA-CNRS-Université Joseph Fourier, 71 avenue des Martyrs, 38044 Grenoble
Cedex 9
IBS / SYNCHROTRON Groupe Synchrotron, Institut de Biologie Structurale J. P. Ebel, UMR 5075 CEACNRS-Université Joseph Fourier, 71 avenue des Martyrs, 38044 Grenoble Cedex 9
ISTerre
Institut des Sciences de la Terre, Université Joseph Fourier, BP 53, 38041, Grenoble
DCM / CIRE
Equipe Chimie Inorganique Rédox, Département de Chimie Moléculaire, 301 rue de
la Chimie, 38041 Grenoble Cedex 9
DCM / I2BM
Equipe Ingénieurie et Intéractions Biomoléculaires, Département de Chimie
Moléculaire, 301 rue de la Chimie, 38041 Grenoble Cedex 9
SCIB / RICC
Laboratoire de Reconnaissance Ionique et Chimie de Coordination, Service de
Chimie Inorganique et Biologique, UMR_E 3 CEA UJF, FRE CNRS 3200, INAC,
CEA-Grenoble, 17 avenue des Martyrs, 38054 Grenoble cedex 9
LCBM / BIOCAT
Equipe Biocatalyse, Laboratoire de Chimie et Biologie des Métaux, UMR 5249
CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble
LCBM / PMB
Equipe Physico-chimie des Métaux en Biologie, Laboratoire de Chimie et Biologie
des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054
Grenoble
LCBM / BIOCE
Equipe Catalyse Bioinorganique et Environnementale, Laboratoire de Chimie et
Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054
Grenoble
LCBM / BIOMET
Equipe Biologie des Métaux, Laboratoire de Chimie et Biologie des Métaux, UMR
5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble
LCBM / MCT
Equipe Modélisation et Chimie Théorique, Laboratoire de Chimie et Biologie des
Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble
PCV / PHYCHLORO
Equipe Régulation de la dynamique du protéome, de la biogenèse et de la physiologie
du chloroplaste, Laboratoire Physiologie Cellulaire et Végétale, UMR 5168 CEACNRS-INRA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble
IAB
Institut Albert Bonniot, CRI INSERM-UJF U823, Rond-point de la Chantourne,
38706 La Tronche Cedex
EMBL / UVHCI
Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 6 Rue Jules
Horowitz, 38042 Grenoble
63
CHU / BHN
Equipe Biochimie Hormonale et Nutritionnelle, Institut de Département de
Biochimie Toxicologie et Pharmacologie, CHU Grenoble, Hôpital A. Michallon,
CHU / DACP
Département d’Anatomie et de Cytologie Pathologiques, Institut de Département de
Biochimie Toxicologie et Pharmacologie, CHU Grenoble, Hôpital A. Michallon,
64

livret final - Institut des Métaux en Biologie de Grenoble

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