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4. Structure‐based insights into the mechanism of [4Fe‐4S]‐dependent sulfur insertase LarE.

Author

Zecchin, Paolo, Pecqueur, Ludovic, Oltmanns, Jonathan, Velours, Christophe, Schünemann, Volker, Fontecave, Marc, and Golinelli‐Pimpaneau, Béatrice

Abstract

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP‐dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe‐4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster‐free (apo‐) and cluster‐bound (holo‐) forms. The structure of holo‐MmLarE shows that the [4Fe‐4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein‐bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo‐MmLarE and the previously reported biochemical investigations of holo‐LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe‐4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe‐5S] intermediate, and transfers it to the substrate, as in the case of [4Fe‐4S]‐dependent tRNA thiolation enzymes. [ABSTRACT FROM AUTHOR]

Published
2024
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5. Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin

Author

Gervason, Sylvain, Larkem, Djabir, Mansour, Amir Ben, Botzanowski, Thomas, Müller, Christina S., Pecqueur, Ludovic, Le Pavec, Gwenaelle, Delaunay-Moisan, Agnès, Brun, Omar, Agramunt, Jordi, Grandas, Anna, Fontecave, Marc, Schünemann, Volker, Cianférani, Sarah, Sizun, Christina, Tolédano, Michel B., and D’Autréaux, Benoit

Published
2019
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12. Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes

Author

Faivre, Bruno, primary, Lombard, Murielle, additional, Fakroun, Soufyan, additional, Vo, Chau-Duy-Tam, additional, Goyenvalle, Catherine, additional, Guérineau, Vincent, additional, Pecqueur, Ludovic, additional, Fontecave, Marc, additional, De Crécy-Lagard, Valérie, additional, Brégeon, Damien, additional, and Hamdane, Djemel, additional

Published
2021
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13. Structural Evidence for a [4Fe‐5S] Intermediate in the Non‐Redox Desulfuration of Thiouracil

Author

Zhou, Jingjing, primary, Pecqueur, Ludovic, additional, Aučynaitė, Agota, additional, Fuchs, Jonathan, additional, Rutkienė, Rasa, additional, Vaitekūnas, Justas, additional, Meškys, Rolandas, additional, Boll, Matthias, additional, Fontecave, Marc, additional, Urbonavičius, Jaunius, additional, and Golinelli‐Pimpaneau, Béatrice, additional

Published
2020
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14. Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion

Author

Valencia-Gallardo, Cesar, Bou-Nader, Charles, Aguilar-Salvador, Daniel-Isui, Carayol, Nathalie, Quenech’Du, Nicole, Pecqueur, Ludovic, Park, HaJeung, Fontecave, Marc, Izard, Tina, Tran Van Nhieu, Guy, Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie des Processus Biologiques (LCPB), Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Chaire Chimie des processus biologiques, and Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

15. The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core

Author

Rajasekar, Karthik V., Zdanowski, Konrad, Yan, Jun, Hopper, Jonathan T. S., Francis, Marie-Louise R., Seepersad, Colin, Sharp, Connor, Pecqueur, Ludovic, Werner, Jörn M., Robinson, Carol V., Mohammed, Shabaz, Potts, Jennifer R., and Kleanthous, Colin

Subjects
Magnetic Resonance Spectroscopy, Science, Sigma Factor, Article, Kinetics, Oxidative Stress, Zinc, Bacterial Proteins, Amino Acid Sequence, Cysteine, Hydrophobic and Hydrophilic Interactions, Oxidation-Reduction
Abstract

Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σR preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA–σR complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all σR-binding residues are sequestered back into its hydrophobic core, releasing σR to activate transcription of anti-oxidant genes., Counteracting oxidative stress is essential in all organisms. Here, the authors outline a mechanism used by actinomycete bacteria in which oxidation of zinc-binding RsrA blocks its interaction with σR by sequestering hydrophobic residues used to bind σR within its own core.

Published
2016

18. Bioinspired Artificial [FeFe]-Hydrogenase with a Synthetic H-Cluster

Author

Papini, Cecilia, primary, Sommer, Constanze, additional, Pecqueur, Ludovic, additional, Pramanik, Debajyoti, additional, Roy, Souvik, additional, Reijerse, Edward J., additional, Wittkamp, Florian, additional, Artero, Vincent, additional, Lubitz, Wolfgang, additional, and Fontecave, Marc, additional

Published
2019
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20. Structural Evidence for a [4Fe‐5S] Intermediate in the Non‐Redox Desulfuration of Thiouracil.

Author

Zhou, Jingjing, Pecqueur, Ludovic, Aučynaitė, Agota, Fuchs, Jonathan, Rutkienė, Rasa, Vaitekūnas, Justas, Meškys, Rolandas, Boll, Matthias, Fontecave, Marc, Urbonavičius, Jaunius, and Golinelli‐Pimpaneau, Béatrice

Subjects
*THIOURACIL, *DESULFURIZATION, *SITE-specific mutagenesis, *CRYSTAL structure, *SULFUR
Abstract

We recently discovered a [Fe‐S]‐containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Å resolution is reported; it harbors a [4Fe‐4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4‐thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non‐protein‐bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe‐5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site‐directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non‐redox enzymatic desulfuration of thiouracil, in which a [4Fe‐4S] cluster binds and activates the sulfur atom of the substrate. [ABSTRACT FROM AUTHOR]

Published
2021
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24. Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O2Resistance and Catalytic Bias?

Author

Caserta, Giorgio, primary, Papini, Cecilia, additional, Adamska-Venkatesh, Agnieszka, additional, Pecqueur, Ludovic, additional, Sommer, Constanze, additional, Reijerse, Edward, additional, Lubitz, Wolfgang, additional, Gauquelin, Charles, additional, Meynial-Salles, Isabelle, additional, Pramanik, Debajyoti, additional, Artero, Vincent, additional, Atta, Mohamed, additional, del Barrio, Melisa, additional, Faivre, Bruno, additional, Fourmond, Vincent, additional, Léger, Christophe, additional, and Fontecave, Marc, additional

Published
2018
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25. Reversible redox-and zinc-dependent dimerization of the Escherichia coli Fur protein

Author

D'Autreaux, Benoit, Pecqueur, Ludovic, De Peredo, Anne Gonzalez, Diederix, Rutger E.M., Caux-Thang, Christelle, Tabet, Lyes, Bersch, Beate, Forest, Eric, and Michaud-Soret, Isabelle

Subjects
Escherichia coli -- Physiological aspects, Oxidation-reduction reaction -- Analysis, Biological sciences, Chemistry
Abstract

The equilibria that exist between dimeric Escherichia coli Fur and higher oligomers is described. It is established that to hold E. coli Fur in a dimeric state, coordination of the zinc ion and the redox state of the cysteines are essential, and oxidizing reagents, diamide, disrupt the dimeric structure leading to monomer formation.

Published
2007

30. An extended dsRBD is required for post-transcriptional modification in human tRNAs

Author

Bou-Nader, Charles, Pecqueur, Ludovic, Bregeon, Damien, Kamah, Amina, Guérineau, Vincent, Golinelli-Pimpaneau, Béatrice, Guimarães, Beatriz G., Fontecave, Marc, Hamdane, Djemel, Laboratoire de Chimie des Processus Biologiques (LCPB), Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF)-Université Pierre et Marie Curie - Paris 6 (UPMC), Traduction eucaryote (TE), Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Chaire Chimie des processus biologiques, Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Collège de France - Chaire Chimie des processus biologiques, Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), HAL-UPMC, Gestionnaire, Centre National de la Recherche Scientifique (CNRS) - Collège de France - Université Pierre et Marie Curie - Paris 6 (UPMC), Adaptation Biologique et Vieillissement (B2A), Université Pierre et Marie Curie - Paris 6 (UPMC) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Institut fédératif de recherches 147, CNRS UMR 8576, Université Lille-Nord, Synchroton SOLEIL, and Université de Lille-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Binding Sites, Saccharomyces cerevisiae Proteins, Nucleic Acid Enzymes, Flavin Mononucleotide, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], RNA, Transfer, Catalytic Domain, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [CHIM] Chemical Sciences, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], RNA Processing, Post-Transcriptional, Oxidoreductases, Oxidation-Reduction, Protein Binding
Abstract

International audience; In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upreg-ulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved ␣-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast comple-mentation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the ␣-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs.

Published
2015

31. Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin

Author

Ahmad, Shoeb, Pecqueur, Ludovic, Dreier, Birgit, Hamdane, Djemel, Aumont-Nicaise, Magali, Plückthun, Andreas, Knossow, Marcel, Gigant, Benoît, Ahmad, Shoeb, Pecqueur, Ludovic, Dreier, Birgit, Hamdane, Djemel, Aumont-Nicaise, Magali, Plückthun, Andreas, Knossow, Marcel, and Gigant, Benoît

Abstract

Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.

Published
2016

33. Role of the zinc and the cysteines in the dimerization of the Ferric Uptake Regulator (FUR) from E. coli : a structural approach using NMR

Author

Pecqueur, Ludovic, Pecqueur, Ludovic, Laboratoire de Physico-chimie des Métaux en Biologie (LPCMB), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph-Fourier - Grenoble I, Michaud-Soret Isabelle(imichaud@cea.fr), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
cystéines, anisotropie de fluorescence, zinc, cristallographie, fluorescence anisotropy, dichroïsme circulaire, RMN, NMR, circular dichroism, Ferric Uptake Regulator, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Dimerization, cristallography, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], FUR, Dimérisation
Abstract

The FUR protein (Ferric Uptake Regulator) is a global regulator ubiquitous in Gram-negative bacteria. Binding of Fe2+ in vivo activates the FUR protein for DNA binding. Fe-FUR binds to the promotor of FUR regulated genes, causing their repression. This work consists of an NMR structural study of the non-activated dimer of FUR (2*17 kDa) which contains one zinc ion per monomer. The FUR monomer was also studied and does not contain the zinc ion. Reduction of the cysteines, and binding of zinc to the protein, leads to dimerization. Circular dichroism and NMR showed that folding of the C-terminal domain of the monomer (dimerization domain) occurs upon dimerization. The secondary structure observed in the monomer and the dimer are similar, except for the first residues. In the monomer, these are organized as a helix while they are unfolded in the non-activated dimer. As this first helix is observed in the activated dimer of P.aeruginosa, we propose it plays a role in the regulation mechanism. A truncated protein named FUR1-82 was constructed, purified, and crystallized. Its structure is superimposable onto the N-terminal domain structure of P. aeruginosa FUR and the 1H-15N-HSQC spectra of FUR1-82 and FUR monomer are nearly identical. Binding of the monomer and the dimer to the DNA consensus sequence study, using fluorescence anisotropy, showed that both forms are able to bind DNA specifically, but with different affinities. The monomer has a 5 fold decreased affinity compared to the dimer in presence of excess metal. Overall, the results obtained allowed us to propose a dimerization mechanism of E.coli FUR and an activation mechanism with a role for the N-terminal Α helix., La protéine FUR (Ferric Uptake Regulator) est un régulateur global ubiquitaire chez les bactéries Gram-négatives. Sa liaison au Fe2+, in vivo, entraîne la répression de l'expression des gènes qu'elle contrôle. Ce travail est une étude structurale par RMN de la forme dimérique non activée de FUR d'Escherichia coli, un dimère de 2*17 kDa contenant un ion zinc par monomère. Une forme monomérique oxydée, capable de dimériser en présence de réducteur et de zinc, a également été isolée et étudiée. Le dichroïsme circulaire et la RMN montrent que la dimérisation entraîne une structuration du domaine C-terminal lors de l'incorporation du zinc. Les structures secondaires du domaine N-terminal du monomère et du dimère sont très proches. Seuls les premiers résidus sont structurés en hélice Α dans le monomère et déstructurés dans le dimère non activé. Cette hélice, observée dans le dimère activé de FUR de P. aeruginosa, pourrait jouer un rôle dans le mécanisme de régulation. Une protéine tronquée (FUR1-82) a été construite, purifiée, cristallisée. Sa structure est superposable à celle du domaine N-terminal de FUR de P. aeruginosa et le spectre 1H-15N-HSQC est superposable aux signaux du domaine N-terminal de FUR monomère. L'étude, par anisotropie de fluorescence, de la liaison du monomère et du dimère à l'ADN montre qu'ils se lient spécifiquement à l'ADN en présence de métal, contrairement à la forme tronquée. L'affinité du monomère pour l'ADN est 5 fois plus faible que celle du dimère en excès de métal. L'ensemble de ces données nous a permis de proposer un mécanisme de dimérisation de FUR d'E. coli ainsi qu'un mécanisme d'activation mettant en jeu cette hélice Α N-terminale.

Published
2005

34. From Enzyme Maturation to Synthetic Chemistry : The Case of Hydrogenases

Author

Artero, Vincent, Berggren, Gustav, Atta, Mohamed, Caserta, Giorgio, Roy, Souvik, Pecqueur, Ludovic, Fontecave, Marc, Artero, Vincent, Berggren, Gustav, Atta, Mohamed, Caserta, Giorgio, Roy, Souvik, Pecqueur, Ludovic, and Fontecave, Marc

Abstract

CONSPECTUS: Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases. In this Account, we first briefly describe the structure of the unique dinuclear organometallic active sites of the two classes of hydrogenases as well as the complex protein machineries involved in their biosynthesis, their so-called maturation processes. This knowledge allows for the development of a fruitful bioinspired chemistry approach, which has already led to a number of interesting and original catalysts mimicking the natural active sites. More specifically, we describe our own attempts to prepare artificial hydrogenases. This can be achieved via the standard bioinspired approach using the combination of a synthetic bioinspired catalyst and a polypeptide scaffold. Such hybrid complexes provide the opportunity to optimize the system by manipulating both the catalyst through chemical synthesis and the protein component through mutagenesis. We also raise the possibility to reach such artificial systems via an original strategy based on mimicking the enzyme maturation pathways. This is illustrated in this Account by two examples developed in our laboratory. First, we show how the preparation of a lysozyme-{Mn-I(CO)(3)} hybrid and its clean reaction with a nickel complex led us to generate a new class of binuclear Ni-Mn H-2-evolving catalysts mimicking the active site of [NiFe]-hydrogenases. Then we describe how we were able to rationally de

Published
2015
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35. A new gene involved in coenzyme Q biosynthesis in Escherichia coli: UbiI functions in aerobic C5-hydroxylation

Author

Hajj Chehade, Mahmoud, Loiseau, Laurent, Lombard, Murielle, Pecqueur, Ludovic, Ismail, Alexandre, Smadja, Myriam, Golinelli-Pimpaneau, Béatrice, Mellot-Draznieks, Caroline, Hamelin, Olivier, Aussel, Laurent, Kieffer-Jaquinod, Sylvie, Labessan, Natty, Barras, Frédéric, Fontecave, Marc, Pierrel, Fabien, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Chaire Chimie des processus biologiques, Laboratoire de Chimie des Processus Biologiques (LCPB), Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Collège de France - Chaire Chimie des processus biologiques, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
MESH: Mutagenesis, Site-Directed, MESH: Binding Sites, MESH: Escherichia coli, MESH: Flavin-Adenine Dinucleotide, MESH: Aerobiosis, MESH: Hydroxylation, MESH: Escherichia coli Proteins, MESH: Ubiquinone, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Mixed Function Oxygenases, MESH: Hydrolases
Abstract

International audience; Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.

Published
2013

36. Structure of a kinesin-tubulin complex and implications for kinesin motility

Author

Gigant, Benoît, Wang, Weiyi, Dreier, Birgit, Jiang, Qiyang, Pecqueur, Ludovic, Plückthun, Andreas, Wang, Chunguang, Knossow, Marcel, University of Zurich, and Wang, Chunguang

Subjects
1315 Structural Biology, 10019 Department of Biochemistry, 1312 Molecular Biology, 570 Life sciences, biology
Published
2013

38. Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase: EQUILIBRIUM BETWEEN INACTIVE HEXAMER AND ACTIVE DIMER

Author

Mouilleron, Stéphane, Badet-Denisot, Marie-Ange, Pecqueur, Ludovic, Madiona, Karine, Assrir, Nadine, Badet, Bernard, Golinelli-Pimpaneau, Béatrice, Institut de Chimie des Substances Naturelles (ICSN), and Centre National de la Recherche Scientifique (CNRS)

Subjects
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing), Models, Molecular, Structure-Activity Relationship, Allosteric Regulation, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Escherichia coli Proteins, Enzyme Stability, Escherichia coli, Enzymology, Protein Multimerization, Protein Structure, Quaternary
Abstract

International audience; The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

Published
2012

41. ubiI, a New Gene in Escherichia coli Coenzyme Q Biosynthesis, Is Involved in Aerobic C5-hydroxylation

Author

Hajj Chehade, Mahmoud, primary, Loiseau, Laurent, additional, Lombard, Murielle, additional, Pecqueur, Ludovic, additional, Ismail, Alexandre, additional, Smadja, Myriam, additional, Golinelli-Pimpaneau, Béatrice, additional, Mellot-Draznieks, Caroline, additional, Hamelin, Olivier, additional, Aussel, Laurent, additional, Kieffer-Jaquinod, Sylvie, additional, Labessan, Natty, additional, Barras, Frédéric, additional, Fontecave, Marc, additional, and Pierrel, Fabien, additional

Published
2013
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44. Chemical assembly of multiple metal cofactors: The heterologously expressed multidomain [FeFe]-hydrogenase from Megasphaera elsdenii

Author

Caserta, Giorgio, Adamska-Venkatesh, Agnieszka, Pecqueur, Ludovic, Atta, Mohamed, Artero, Vincent, Roy, Souvik, Reijerse, Edward, Lubitz, Wolfgang, Fontecave, Marc, Caserta, Giorgio, Adamska-Venkatesh, Agnieszka, Pecqueur, Ludovic, Atta, Mohamed, Artero, Vincent, Roy, Souvik, Reijerse, Edward, Lubitz, Wolfgang, and Fontecave, Marc

Abstract

[FeFe]-hydrogenases are unique and fascinating enzymes catalyzing the reversible reduction of protons into hydrogen. These metalloenzymes display extremely large catalytic reaction rates at very low overpotential values and are, therefore, studied as potential catalysts for bioelectrodes of electrolyzers and fuel cells. Since they contain multiple metal cofactors whose biosynthesis depends on complex protein machineries, their preparation is difficult. As a consequence still few have been purified to homogeneity allowing spectroscopic and structural characterization. As part of a program aiming at getting easy access to new hydrogenases we report here a methodology based on a purely chemical assembly of their metal cofactors. This methodology is applied to the preparation and characterization of the hydrogenase from the fermentative anaerobic rumen bacterium Megasphaera elsdenii, which has only been incompletely characterized in the past.

45. Bioinspired Artificial [FeFe]-Hydrogenase with a Synthetic H-Cluster

Author

Papini, Cecilia, Sommer, Constanze, Pecqueur, Ludovic, Pramanik, Debajyoti, Roy, Souvik, Reijerse, Edward J., Wittkamp, Florian, Artero, Vincent, Lubitz, Wolfgang, Fontecave, Marc, Papini, Cecilia, Sommer, Constanze, Pecqueur, Ludovic, Pramanik, Debajyoti, Roy, Souvik, Reijerse, Edward J., Wittkamp, Florian, Artero, Vincent, Lubitz, Wolfgang, and Fontecave, Marc

Abstract

[FeFe]-hydrogenases are powerful hydrogen evolution biocatalysts because of the activity of their unique metallocofactor, the H-cluster, derived from the association of a [4Fe-4S] cluster and a [2Fe]-subcluster, containing 2 CO and 2 CN– ligands as well as an aza-propanedithiolate (adt2–) ligand bridging the two Fe atoms. Various analogues of the H-cluster have been assembled in the iron–sulfur protein HydF, containing a [4Fe-4S] cluster, by reaction with a series of synthetic di-iron complexes mimicking the [2Fe]-subcluster of hydrogenases. The mimics contain a mixture of CO and CN– ligands and various dichalcogenide (S or Se) bridging ligands. The resulting hybrid proteins have been characterized and shown to behave as active artificial hydrogenases. On the basis of the present structure–activity relationship study, we report that the most active and stable artificial hydrogenase was obtained with the mimic containing a propane diselenol bridging ligand. This opens the possibility to developing a class of hydrogen-evolving enzymes with a synthetic active site mimicking that of the natural hydrogenase.

46. Structural and functional characterization of the hydrogenase-maturation HydF protein

Author

Caserta, Giorgio, Pecqueur, Ludovic, Adamska-Venkatesh, Agnieszka, Papini, Cecilia, Roy, Souvik, Artero, Vincent, Atta, Mohamed, Reijerse, Edward, Lubitz, Wolfgang, Fontecave, Marc, Caserta, Giorgio, Pecqueur, Ludovic, Adamska-Venkatesh, Agnieszka, Papini, Cecilia, Roy, Souvik, Artero, Vincent, Atta, Mohamed, Reijerse, Edward, Lubitz, Wolfgang, and Fontecave, Marc

Abstract

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H+ and H2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN− and azapropanedithiolate (adt2−) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe2(adt)(CO)4(CN)2]2− is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.

47. From Enzyme Maturation to Synthetic Chemistry: The Case of Hydrogenases

Author

Artero, Vincent, Berggren, Gustav, Atta, Mohamed, Caserta, Giorgio, Roy, Souvik, Pecqueur, Ludovic, Fontecave, Marc, Artero, Vincent, Berggren, Gustav, Atta, Mohamed, Caserta, Giorgio, Roy, Souvik, Pecqueur, Ludovic, and Fontecave, Marc

Abstract

Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases. In this Account, we first briefly describe the structure of the unique dinuclear organometallic active sites of the two classes of hydrogenases as well as the complex protein machineries involved in their biosynthesis, their so-called maturation processes. This knowledge allows for the development of a fruitful bioinspired chemistry approach, which has already led to a number of interesting and original catalysts mimicking the natural active sites. More specifically, we describe our own attempts to prepare artificial hydrogenases. This can be achieved via the standard bioinspired approach using the combination of a synthetic bioinspired catalyst and a polypeptide scaffold. Such hybrid complexes provide the opportunity to optimize the system by manipulating both the catalyst through chemical synthesis and the protein component through mutagenesis. We also raise the possibility to reach such artificial systems via an original strategy based on mimicking the enzyme maturation pathways. This is illustrated in this Account by two examples developed in our laboratory. First, we show how the preparation of a lysozyme–{MnI(CO)3} hybrid and its clean reaction with a nickel complex led us to generate a new class of binuclear Ni-Mn H2-evolving catalysts mimicking the active site of [NiFe]-hydrogenases. Then we describe how we were able to rationally design and prepare

48. ShigellaIpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion

Author

Valencia-Gallardo, Cesar, Bou-Nader, Charles, Aguilar-Salvador, Daniel-Isui, Carayol, Nathalie, Quenech’Du, Nicole, Pecqueur, Ludovic, Park, HaJeung, Fontecave, Marc, Izard, Tina, and Tran Van Nhieu, Guy

Abstract

The Shigellatype III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 Å resolution crystal structure of IpaA VBS3 in complex with the talin H1–H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1–H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.

Published
2019
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49. Structure-based insights into the mechanism of [4Fe-4S]-dependent sulfur insertase LarE.

Author

Zecchin P, Pecqueur L, Oltmanns J, Velours C, Schünemann V, Fontecave M, and Golinelli-Pimpaneau B

Subjects
Ligands, Cysteine chemistry, Catalysis, Sulfur chemistry, Sulfur metabolism, Chlorides metabolism, Iron-Sulfur Proteins chemistry
Abstract

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP-dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe-4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster-free (apo-) and cluster-bound (holo-) forms. The structure of holo-MmLarE shows that the [4Fe-4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein-bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo-MmLarE and the previously reported biochemical investigations of holo-LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe-5S] intermediate, and transfers it to the substrate, as in the case of [4Fe-4S]-dependent tRNA thiolation enzymes., (© 2023 The Protein Society.)

Published
2024
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50. Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion.

Author

Valencia-Gallardo C, Bou-Nader C, Aguilar-Salvador DI, Carayol N, Quenech'Du N, Pecqueur L, Park H, Fontecave M, Izard T, and Tran Van Nhieu G

Subjects
Antigens, Bacterial genetics, Bacterial Proteins genetics, Crystallography, X-Ray, HeLa Cells, Humans, Protein Domains, Pseudopodia genetics, Pseudopodia metabolism, Shigella flexneri genetics, Shigella flexneri metabolism, Static Electricity, Talin genetics, Talin metabolism, Antigens, Bacterial chemistry, Bacterial Proteins chemistry, Cell Adhesion, Pseudopodia chemistry, Shigella flexneri chemistry, Talin chemistry
Abstract

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 Å resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2019
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