Author: "Ekaterina Darii" - Searchworks@Jio Institute Digital Library Search Results

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28 results on '"Ekaterina Darii"'

1. Simplified in Vitro and in Vivo Bioaccess to Prenylated Compounds

Author: Julie Couillaud, Juan Rico, Alyssa Rubini, Tarek Hamrouni, Elise Courvoisier-Dezord, Jean-Louis Petit, Aline Mariage, Ekaterina Darii, Katia Duquesne, Véronique de Berardinis, and Gilles Iacazio
Subjects: Chemistry, QD1-999
Published: 2019
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2. Exploring natural biodiversity to expand access to microbial terpene synthesis

Author: Juan Rico, Katia Duquesne, Jean-Louis Petit, Aline Mariage, Ekaterina Darii, Frédéric Peruch, Véronique de Berardinis, and Gilles Iacazio
Subjects: IPK, Terpene biosynthesis, Neurosporene, Microbial production, Microbiology, QR1-502
Abstract: Abstract Background Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established—mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes. Results The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule. Conclusion IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.
Published: 2019
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3. Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations

Author: Sophia Belkhelfa, David Roche, Ivan Dubois, Anne Berger, Valérie A. Delmas, Laurence Cattolico, Alain Perret, Karine Labadie, Aude C. Perdereau, Ekaterina Darii, Emilie Pateau, Véronique de Berardinis, Marcel Salanoubat, Madeleine Bouzon, and Volker Döring
Subjects: methylotrophic bacteria, directed evolution, continuous culture, genomics, transcriptomics, lactate, Microbiology, QR1-502
Abstract: The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.
Published: 2019
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4. Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes

Author: Nadia Perchat, Christelle Dubois, Rémi Mor-Gautier, Sophie Duquesne, Christophe Lechaplais, David Roche, Stéphanie Fouteau, Ekaterina Darii, Alain Perret, Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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), Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Commissariat à l'Energie Atomique et aux Energies Alternatives, Centre National de la Recherche Scientifique (CNRS)European Commission, Université Evry-Val-d'Essonne/Université Paris Saclay, Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Acinetobacter, [SDV]Life Sciences [q-bio], Escherichia coli, beta-Alanine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell Biology, Molecular Biology, Biochemistry, Metabolic Networks and Pathways, Transaminases, Biosynthetic Pathways
Abstract: International audience; Bacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these en-zymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for L- aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of beta-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 delta panD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes.
Published: 2022

5. First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures

Author: Yves Gimbert, Sandra Alves, François Fenaille, Ekaterina Darii, Alain Perret, Amina S. Woods, Jean-Claude Tabet, Annelaure Damont, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Département de Chimie Moléculaire (DCM), Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Chimie Structurale Organique et Biologique (CSOB), Institut Parisien de Chimie Moléculaire (IPCM), Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), DMTS, Université Paris Saclay, CEA, INRAE, SPI, Synthèse, Structure et Fonction de Molécules Bioactives (SSFMB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), Service de Pharmacologie et Immunoanalyse (SPI), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Subjects: chemistry.chemical_classification, Hydride, Electrospray ionization, 010401 analytical chemistry, 010402 general chemistry, 01 natural sciences, Dissociation (chemistry), 0104 chemical sciences, chemistry.chemical_compound, Crystallography, chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Structural Biology, Molecule, Non-covalent interactions, Isotopologue, Carboxylate, Isomerization, ComputingMilieux_MISCELLANEOUS, Spectroscopy
Abstract: International audience; Mass spectrometric investigations of noncovalent binding between low molecular weight compounds revealed the existence of gas-phase (GP) noncovalent complex (NCC) ions involving zwitterionic structures. ESI MS is used to prove the formation of stable sodiated NCC anions between fructose (F6P) and arginine (R) moieties. Theoretical calculations indicate a folded solvated salt (i.e., sodiated carboxylate interacting with phosphate) rather than a charge-solvated form. Under standard CID conditions, [(F6P+R-H+Na)-H]− competitively forms two major product ions (PIs) through partner splitting [(R-H+Na) loss] and charge-induced cross-ring cleavage while preserving the noncovalent interactions (noncovalent product ions (NCPIs)). MS/MS experiments combined with in-solution proton/deuteron exchanges (HDXs) demonstrated an unexpected labeling of PIs, i.e., a correlated D-enrichment/D-depletion. An increase in activation time up to 3000 ms favors such processes when limited to two H/D exchanges. These results are rationalized by interpartner hydride/deuteride exchanges (⟨HDX⟩) through stepwise isomerization/dissociation of sodiated NCC-d11 anions. In addition, the D-enrichment/D-depletion discrepancy is further explained by back HDX with residual water in LTQ (selective for the isotopologue NCPIs as shown by PI relaxation experiments). Each isotopologue leads to only one back HDX unlike multiple HDXs generally observed in GP. This behavior shows that NCPIs are zwitterions with charges solvated by a single water molecule, thus generating a back HDX through a relay mechanism, which quenches the charges and prevents further back HDX. By estimating back HDX impact on D-depletion, the interpartner ⟨HDX⟩ complementarity was thus illustrated. This is the first description of interpartner ⟨HDX⟩ and selective back HDX validating salt-solvated structures.
Published: 2021

6. Simplified in Vitro and in Vivo Bioaccess to Prenylated Compounds

Author: Jean-Louis Petit, Alyssa Rubini, Véronique de Berardinis, Elise Courvoisier-Dezord, Julie Couillaud, Katia Duquesne, Juan Rico, Gilles Iacazio, Ekaterina Darii, Tarek Hamrouni, Aline Mariage, Institut des Sciences Moléculaires de Marseille (ISM2), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), and ANR-14-CE06-0027,Reset,Mini voie de biosynthèse des terpènes(2014)
Subjects: lcsh:Chemistry, Terpene, lcsh:QD1-999, Prenylation, Chemistry, Stereochemistry, In vivo, [SDV]Life Sciences [q-bio], [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], General Chemical Engineering, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, General Chemistry, In vitro
Abstract: All naturally produced terpenes are derived from two universal C5 diphosphate precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Various prenyl transferases use DMAPP ...
Published: 2019

7. 2-Deoxyribose-5-phosphate aldolase, a remarkably tolerant aldolase towards nucleophile substrates

Author: Véronique de Berardinis, Marcel Salanoubat, Ekaterina Darii, Domitille Chambre, Aline Mariage, Jean-Louis Petit, Marielle Lemaire, Christine Guérard-Hélaine, Virgil Hélaine, Institut de Chimie de Clermont-Ferrand (ICCF), SIGMA Clermont (SIGMA Clermont)-Institut de Chimie du CNRS (INC)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Université Clermont Auvergne (UCA), Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS), 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 Paris-Saclay-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Association géologique auboise, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay
Subjects: Stereochemistry, [SDV]Life Sciences [q-bio], Dihydroxyacetone, 010402 general chemistry, Cyclopentanone, Glyceraldehyde 3-Phosphate, 01 natural sciences, Catalysis, Substrate Specificity, chemistry.chemical_compound, Bacterial Proteins, Aldol reaction, Nucleophile, Escherichia coli, Materials Chemistry, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Arthrobacter, ComputingMilieux_MISCELLANEOUS, Aldehyde-Lyases, Aldehydes, Glycolaldehyde, biology, 010405 organic chemistry, Aldolase A, Metals and Alloys, Biodiversity, General Chemistry, biology.organism_classification, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, chemistry, Electrophile, Biocatalysis, Ceramics and Composites, biology.protein, Arthrobacter chlorophenolicus
Abstract: International audience; We explored a collection of 2-deoxyribose-5-phosphate aldolases (DERAs) from biodiversity for their nucleophile substrate promiscuity. The DERAs were screened using as nucleophiles propanone, propanal, cyclobutanone, cyclopentanone, dihydroxyacetone, and glycolaldehyde with L-glyceraldehyde-3-phosphate as an electrophile in aldol addition. A DERA from Arthrobacter chlorophenolicus (DERAArthro) efficiently allowed the synthesis of the corresponding aldol adducts in good yields, displaying complementarity in terms of configuration and substrate specificity with fructose-6-phosphate aldolase, the only previously known aldolase with a large nucleophile tolerance.
Published: 2019

8. Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations

Author: Emilie Pateau, Marcel Salanoubat, Aude Perdereau, Véronique de Berardinis, Valérie Delmas, Laurence Cattolico, Madeleine Bouzon, Ekaterina Darii, Karine Labadie, Alain Perret, Ivan Dubois, Sophia Belkhelfa, David Roche, Anne Berger, Volker Döring, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Centre Interlangues - Texte, Image, Langage (TIL), Université de Bourgogne (UB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Structure et évolution des génomes (SEG), CNS-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique d'Evry (IG), Institut de Biologie François JACOB (JACOB), 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 Paris-Saclay, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Microbiology (medical), methylotrophic bacteria, [SDV]Life Sciences [q-bio], lcsh:QR1-502, Dehydrogenase, 7. Clean energy, Microbiology, lcsh:Microbiology, 03 medical and health sciences, chemistry.chemical_compound, transcriptomics, Ribosomal protein, genomics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, directed evolution, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, lactate, Strain (chemistry), biology, 030306 microbiology, Directed evolution, biology.organism_classification, chemistry, Biochemistry, continuous culture, Methanol, Methylobacterium extorquens, Energy source, Bacteria
Abstract: The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.
Published: 2019

9. Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments

Author: Jean-François Gallard, Jean Weissenbach, Denis Le Paslier, Marion L. Chevallier, Ewen Lescop, Sébastien Chaussonnerie, Ekaterina Darii, Agnès Barbance, Oriane Della-Negra, Stéphane Vuilleumier, Thierry Woignier, Edgardo Ugarte, Delphine Muselet, Florian Lagarde, Nuria Fonknechten, Pierre-Loïc Saaidi, Gwenaël Imfeld, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Institut méditerranéen de biodiversité et d'écologie marine et continentale (IMBE), Avignon Université (AU)-Aix Marseille Université (AMU)-Institut de recherche pour le développement [IRD] : UMR237-Centre National de la Recherche Scientifique (CNRS), Génétique moléculaire, génomique, microbiologie (GMGM), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Hydrologie et de Géochimie de Strasbourg (LHyGeS), Ecole et Observatoire des Sciences de la Terre (EOST), Institut national des sciences de l'Univers (INSU - CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université de Strasbourg (UNISTRA)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Pollution, Insecticides, West Indies, media_common.quotation_subject, Environmental pollution, 010501 environmental sciences, Biology, 01 natural sciences, biodegradation, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Humans, Soil Pollutants, Environmental Chemistry, pollution, Martinique, Ecosystem, 0105 earth and related environmental sciences, media_common, West indies, Aquatic ecosystem, structure elucidation, Musa, General Chemistry, 6. Clean water, Transformation (genetics), transformation product, 13. Climate action, Chlordecone, Environmental chemistry, persistant organic pollutant, Degradation (geology), [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Microcosm, [CHIM.OTHE]Chemical Sciences/Other
Abstract: International audience; Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.
Published: 2019

10. Exploring natural biodiversity to expand access to microbial terpene synthesis

Author: Gilles Iacazio, Aline Mariage, Véronique de Berardinis, Ekaterina Darii, Jean-Louis Petit, Frédéric Peruch, Katia Duquesne, Juan Rico, Institut des Sciences Moléculaires de Marseille (ISM2), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie des Polymères Organiques (LCPO), Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-14-CE06-0027,Reset,Mini voie de biosynthèse des terpènes(2014), 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 d'Évry-Val-d'Essonne (UEVE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)
Subjects: 0106 biological sciences, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, lcsh:QR1-502, Mevalonic Acid, Bioengineering, Erythritol, 01 natural sciences, Applied Microbiology and Biotechnology, lcsh:Microbiology, Terpene, Metabolic engineering, 03 medical and health sciences, chemistry.chemical_compound, Hemiterpenes, Organophosphorus Compounds, Biosynthesis, 010608 biotechnology, Methanococcoides burtonii, Escherichia coli, Microbial production, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Carotenoid, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Bacteria, biology, Terpenes, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Research, Biodiversity, biology.organism_classification, Archaea, Carotenoids, Lycopene, Terpene biosynthesis, Metabolic Engineering, Biochemistry, Batch Cell Culture Techniques, Neurosporene, [CHIM.OTHE]Chemical Sciences/Other, Biotechnology, IPK
Abstract: Background Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established—mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes. Results The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule. Conclusion IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance. Electronic supplementary material The online version of this article (10.1186/s12934-019-1074-4) contains supplementary material, which is available to authorized users.
Published: 2019

11. De novo structure determination of 3-((3-aminopropyl)amino)- 4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1

Author: Alain Perret, Ekaterina Darii, Lucille Stuani, Marion Thomas, Pierre-Loïc Saaidi, Christophe Lechaplais, Emilie Pateau, Jean-Claude Tabet, Marcel Salanoubat, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Synthèse, Structure et Fonction de Molécules Bioactives (SSFMB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Parisien de Chimie Moléculaire (IPCM), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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 d'Évry-Val-d'Essonne (UEVE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)
Subjects: Magnetic Resonance Spectroscopy, Endocrinology, Diabetes and Metabolism, Metabolite, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Microbial metabolism, Parabens, Tandem mass spectrometry, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Metabolomics, 4-Hydroxybenzoic acid, Tandem Mass Spectrometry, Metabolome, 030304 developmental biology, 0303 health sciences, Acinetobacter, 010401 analytical chemistry, Metabolism, 0104 chemical sciences, Metabolic pathway, chemistry
Abstract: International audience; Introduction Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. Objectives This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). Methods We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D-and 2D-NMR (1 H, 13 C, 1 H-1 H-COSY, 1 H-13 C-HSQC, 1 H-13 C-HMBC and 1 H-15 N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. Results We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxy-benzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the mil-limolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. Conclusion Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.
Published: 2019

12. Characterization of l-Carnitine Metabolism in Sinorhizobium meliloti

Author: Marcel Salanoubat, Christophe Lechaplais, Alain Perret, Nadia Perchat, Ekaterina Darii, Pascal Bazire, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and 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 d'Évry-Val-d'Essonne (UEVE)
Subjects: 0106 biological sciences, enzymology, [SDV]Life Sciences [q-bio], Mutant, Fatty acid degradation, 01 natural sciences, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Carnitine, medicine, L-carnitine, Metabolomics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, Biotransformation, 030304 developmental biology, 0303 health sciences, Sinorhizobium meliloti, biology, Catabolism, bacterial metabolism, biology.organism_classification, Aerobiosis, LC-MS, Betaine, Metabolic pathway, chemistry, Biochemistry, orbitrap, trimethylammonium compounds, Multigene Family, functional genomics, Bacteria, Metabolic Networks and Pathways, 010606 plant biology & botany, medicine.drug, Research Article
Abstract: International audience; L-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from ␥-butyrobetaine (GBB) and can be used as a carbon source. L-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of L-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of L-carnitine biosynthesis and degradation in Sinorhizobium meliloti. The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of L-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti. To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of L-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of L-carnitine as a ubiquitous nutrient. IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of L-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.
Published: 2018

13. Asymmetric reductive amination by a wild-type amine dehydrogenase from the thermophilic bacteria Petrotoga mobilis

Author: Véronique de Berardinis, Carine Vergne-Vaxelaire, Virginie Pellouin, Jean-Louis Petit, Marcel Salanoubat, Aline Mariage, Anne Zaparucha, Ekaterina Darii, Ombeline Mayol, Sylvain David, and Adrien Debard
Subjects: chemistry.chemical_classification, Ketone, biology, 010405 organic chemistry, Stereochemistry, Thermophile, Amine dehydrogenase, Substrate (chemistry), 010402 general chemistry, 01 natural sciences, Reductive amination, Catalysis, 0104 chemical sciences, Enzyme, chemistry, biology.protein, Organic chemistry, NAD+ kinase
Abstract: The biocatalytic reductive amination of ketone to chiral amine is one of the most challenging reactions. Using a genome-mining approach, we found proteins catalyzing the reductive amination of ketones without a carboxylic function in the α or β position. The synthesis of (4S)-4-aminopentanoic acid (ee ≥99.5%) was achieved with the thermoactive amine dehydrogenase (AmDH) AmDH4 from Petrotoga mobilis in 88% yield. The high stability and substrate tolerance make AmDH4 a very good starting point for further discovery of reductive amination biocatalysts with an enlarged substrate range. This is the first report of wild-type enzymes with related genes having proper NAD(P)H–AmDH activity.
Published: 2016

14. Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites

Author: Alexandra Gimbernat, Cécile Fischer, Véronique de Berardinis, Pierre-Loïc Saaidi, Christine Pellé, Jean-Louis Petit, Nadia Perchat, Marielle Besnard-Gonnet, Marcel Salanoubat, Ekaterina Darii, Alain Perret, Maeva Dupont, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), and 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 d'Évry-Val-d'Essonne (UEVE)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], Hypothetical protein, Dehydrogenase, Mass Spectrometry, Hydroxylation, 03 medical and health sciences, chemistry.chemical_compound, Alkaloids, Trigonelline, Hydrolase, 2. Zero hunger, chemistry.chemical_classification, Multidisciplinary, 030102 biochemistry & molecular biology, Acinetobacter, Methylamine, Catabolism, Molecular Sequence Annotation, 030104 developmental biology, Enzyme, chemistry, Biochemistry, PNAS Plus, Multigene Family, Genome, Bacterial, Chromatography, Liquid
Abstract: International audience; Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available. bacterial metabolism | functional genomics | LC/MS-Orbitrap | trigonelline | N-heterocycle degradation E xtensive and accurate bacterial genome annotation is critical for developing a comprehensive and detailed understanding of cellular physiology, and is therefore a major concern in biological research. As a result, 30-40% of genes of a typical ge-nome remain unannotated or associated with a putative function (1, 2). In many cases, function is extrapolated from a small number of characterized proteins (3). In this context, the need for a global effort of experimental assignation, validation, or correction of function is major. Experimental work guided by bioinformatics has proved to be an invaluable tool for assigning new functions (4-7). However, investigations on catabolic pathways are still often conducted in a few model organisms and with a restricted set of nutrients. Given the vast array of natural secondary metabolites and their underrepresentation in metabolic maps, the full range of transformations afforded by bacteria is clearly underestimated. Thus, simply varying the range of organisms tested and the set of nutrients remains a useful tactic for elucidating hidden latent microbial catabolic pathways and providing access to gene function (8-10). Trigonelline (TG; N-methylnicotinate) is a metabolite of nic-otinamide involved in plant cell cycle regulation and oxidative stress (11). Released by legume roots and seeds, such as in alfalfa , it activates nodulation genes in Rhizobium meliloti (12). It is one of the most widely distributed betaines in higher plants (12, 13), and it is also present with different concentration ranges in organisms such as reef-building corals (14, 15), algae (16), and marine plankton, in which it can reach the millimolar range (17, 18). It is likely released in the environment through the death of these organisms. As a consequence, numerous heterotrophic prokaryotes probably use this compound as a nutrient. Surprisingly , the soil bacterium R. meliloti RCR2011 is the only organism reported to use TG as a carbon, nitrogen, and energy source (19). Although an inducible genetic region involved in the degradation of this compound was specified (20, 21), investigations aimed at deciphering this metabolic pathway did not go further. Therefore, the complete set of genes and catabolites involved in this process is not reported. Acinetobacter baylyi ADP1 (ADP1) is a nutritionally versatile strictly aerobic bacterium capable of metabolizing a wide range of aromatic compounds (22). Its extraordinary competence for natural transformation and the ease with which it can be genetically engineered (23, 24) make ADP1 a key organism for the study of soil bacteria metabolism of natural compounds. In this work, we show that ADP1 can use TG as the sole source of carbon, nitrogen, and energy. A gene cluster responsible for TG degradation, which we called tgn (for trigonelline), was revealed Significance The experimental dissection of novel metabolic pathways, from genes and enzymes to metabolites, is a key issue for improving our knowledge of the enzymatic capabilities of the microbial world and providing accurate functional annotation of genomes. We used an integrative methodology combining the phenotyping of a complete genome-scale mutant collection of Acinetobacter baylyi ADP1 with an untargeted liquid chromatography/MS-based approach to uncover the degradation pathway of trigonelline (TG), a widespread osmolyte. We provide extensive information about this unusual N-heterocyclic aromatic degradation route that expands the metabolite repertoire. The occurrence of conserved gene clusters for TG dis-similation in soil, plant-associated, and marine bacteria underlines its environmental abundance.
Published: 2018

15. Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1- phosphate Aldolases

Author: Véronique de Berardinis, Tibor Liptaj, Aline Mariage, Christine Guérard-Hélaine, Marielle Lemaire, Victor Laurent, Angelina Aujon, Marine Debacker, Ekaterina Darii, Mounir Traïkia, Jean-Louis Petit, Marcel Salanoubat, Virgil Hélaine, Martin Breza, Lionel Nauton, Institut de Chimie de Clermont-Ferrand (ICCF), SIGMA Clermont (SIGMA Clermont)-Institut de Chimie du CNRS (INC)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), SIGMA Clermont (SIGMA Clermont), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Institut de Biologie François JACOB (JACOB), 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), Slovak University of Technology, Faculty of Chemical and Food Technology, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), SIGMA Clermont (SIGMA Clermont)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay
Subjects: Stereochemistry, [SDV]Life Sciences [q-bio], 010402 general chemistry, 01 natural sciences, Catalysis, Substrate Specificity, chemistry.chemical_compound, Nucleophile, Aldol reaction, DHAP, Moiety, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Aldehyde-Lyases, Dihydroxyacetone phosphate, Molecular Structure, biology, Chemistry, 010405 organic chemistry, Aldolase A, Stereoisomerism, General Chemistry, General Medicine, Ketones, 0104 chemical sciences, Bacteroides thetaiotaomicron, Dihydroxyacetone Phosphate, Electrophile, biology.protein, Stereoselectivity, Sugars
Abstract: International audience; Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several a-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a ter-tiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new CÀC bond-forming enzyme for stereoselective synthesis of tertiary alcohols.
Published: 2018

16. Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Author: Véronique de Berardinis, Ekaterina Darii, Aurélie Fossey-Jouenne, Marcel Salanoubat, Adrien Debard, Jean-Louis Petit, Marielle Lemaire, Stessy Nepert, Virginie Pellouin, Aline Mariage, Christine Guérard-Hélaine, Anne Zaparucha, Carine Vergne-Vaxelaire, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Clermont-Ferrand (ICCF), SIGMA Clermont (SIGMA Clermont)-Institut de Chimie du CNRS (INC)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), 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 Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Institut de Chimie de Clermont-Ferrand - Clermont Auvergne (ICCF), Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne (UCA)-Sigma CLERMONT (Sigma CLERMONT), 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 d'Évry-Val-d'Essonne (UEVE), Association géologique auboise, Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Sigma CLERMONT (Sigma CLERMONT)-Université Clermont Auvergne (UCA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Thermotolerance, Phosphorylated sugars, Streptococcus mitis, Applied Microbiology and Biotechnology, Fructokinase, Substrate Specificity, Fructokinases, 03 medical and health sciences, chemistry.chemical_compound, Hexokinase, Monosaccharide, [CHIM]Chemical Sciences, Phosphorylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], chemistry.chemical_classification, biology, Kinase, Temperature, ROK family, General Medicine, biology.organism_classification, Promiscuity, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Open reading frame, 030104 developmental biology, Enzyme, chemistry, Biochemistry, Sugars, Biotechnology
Abstract: International audience; Most of the Brepressor, open reading frame, kinase^(ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrK mitis) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 10 6 s −1 M −1 and 2.7 10 5 s −1 M −1 , respectively, but also significant ones toward D-tagatose (3.5 10 2 s −1 M −1) and the unnatural monosaccharides D-altrose (1.1 10 4 s −1 M −1) and D-talose (3.4 10 2 s −1 M −1). Specific activities measured for all hexoses showed a high stereopreference for D-over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermo-tolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70°C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.
Published: 2018

17. Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution

Author: Oren Yishai, Arren Bar-Even, Volker Döring, Ekaterina Darii, Madeleine Bouzon, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE), Max Planck Institute of Molecular Physiology, Max-Planck-Gesellschaft, H2020, grant agreement No 763911, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), and European Project: 763911,eForFuel
Subjects: 0301 basic medicine, synthetic metabolism, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Formates, Microorganism, 030106 microbiology, Biomedical Engineering, glycine cleavage complex, Industrial biotechnology, medicine.disease_cause, Biochemistry, Genetics and Molecular Biology (miscellaneous), Serine, 03 medical and health sciences, chemistry.chemical_compound, Carbon assimilation, Tandem Mass Spectrometry, medicine, Escherichia coli, Formate, directed evolution, Chromatography, High Pressure Liquid, formate assimilation, Carbon Isotopes, Chemistry, General Medicine, Carbon Dioxide, Directed evolution, one-carbon metabolism, Combinatorial chemistry, Glutathione, Carbon, 030104 developmental biology, long-term continuous culture, Directed Molecular Evolution, Adaptive evolution
Abstract: International audience; Endowing biotechnological platform organismswith new carbon assimilation pathways is a key challenge forindustrial biotechnology. Here we report progress toward theconstruction of formatotrophic Escherichia coli strains. Glycineand serine, universal precursors of one-carbon compoundsoxidized during heterotrophic growth, are produced fromformate and CO2 through a reductive route. An adaptiveevolution strategy was applied to optimize the enzymatic steps ofthis route in appropriate selection strains. Metabolic labelingexperiments with 13C-formate confirm the redirected carbonflow.These results demonstrate the high plasticity of the centralcarbon metabolism of E. coli and the applicative potential ofdirected evolution for implementing synthetic pathways inmicroorganisms.
Published: 2018
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18. Meaning and consequence of the coexistence of competitive hydrogen bond/salt forms on the dissociation orientation of non-covalent complexes

Author: Jean-Claude Tabet, Ekaterina Darii, Yves Gimbert, Alain Perret, Sandra Alves, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Département de Chimie Moléculaire - Synthèse Et Réactivité en Chimie Organique (DCM - SeRCO ), Département de Chimie Moléculaire (DCM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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]), Chimie Structurale Organique et Biologique (CSOB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de Génotypage (CNG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Models, Molecular, Spectrometry, Mass, Electrospray Ionization, Clinical Biochemistry, Ionic bonding, Glucose-6-Phosphate, Protonation, 010402 general chemistry, Arginine, 01 natural sciences, Biochemistry, Dissociation (chemistry), Analytical Chemistry, chemistry.chemical_compound, Deprotonation, Isomerism, Tandem Mass Spectrometry, [CHIM]Chemical Sciences, Guanidine, ComputingMilieux_MISCELLANEOUS, Chromatography, Hydrogen bond, 010401 analytical chemistry, Fructosephosphates, Glucosephosphates, Hydrogen Bonding, Cell Biology, General Medicine, 0104 chemical sciences, chemistry, Covalent bond, Zwitterion, Thermodynamics
Abstract: Non-covalent complexes (NCC) between hexose monophosphates (HexP) and arginine (R) were analyzed using ESI MS and MS/MS in negative mode under different (hard, HC and soft, SC) desolvation conditions. High resolution mass spectrometry (HRMS) revealed the presence of different ionic species, namely, homo- and heteromultimers of R and HexP. Deprotonated heterodimers and corresponding sodiated species were enhanced under HC likely due to a decrease in available charge number associated with the reduction of H + /Na + exchange. The quantum calculations showed that the formation of covalent systems is very little exothermic, therefore, such systems are disfavored. Desolvation dependent CID spectra of deprotonated [(HexP+R)‒H] − complexes demonstrated that they can exist within the hydrogen bond (HB) and salt bridge (SB) forms, yielding either NCC separation or covalent bond cleavages, respectively. Although HB forms are the main species, they cannot survive under HC; therefore, the minor SB forms became detectable. Energy-resolved mass spectrometry (ERMS) experiments revealed diagnostic fragment ions from both SB and HB forms, providing evidence that these isomeric forms are inconvertible. SB formation should result from the ionic interactions of highly acidic group of HexP with strongly basic guanidine group of arginine and thus requires an arginine zwitterion (ZW) form. This was confirmed by quantum calculations. Ion-ion interactions are significantly affected by the presence of sodium cation as demonstrated by the fragmentation patterns of sodiated complex species. Regarding CID data, only SB between protonated amino group of R and deprotonated phosphate group of HexP could be suggested, but the primary amine is not enough basic then, the SB must be fleeting. Nevertheless, the observation of the covalent bond cleavages suggests the presence of structures with a free negative charge able to induce fragmentations. Indeed, according to quantum calculations, solvated salt (SS) systems involving Na + /COO − salt solvated by neutral phosphate and negative charge on sugar ring are preferentially formed.
Published: 2017

19. Expanding the reaction space of aldolases using hydroxypyruvate as a nucleophilic substrate

Author: Nicolas Poupard, Thierry Gefflaut, Christine Guérard-Hélaine, Virgil Hélaine, Ekaterina Darii, Jean-Louis Petit, Véronique de Berardinis, Mark Stam, Marcel Salanoubat, Aline Mariage, Marielle Lemaire, Karine Bastard, Israel Sánchez-Moreno, Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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), Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Clermont-Ferrand (ICCF), SIGMA Clermont (SIGMA Clermont)-Institut de Chimie du CNRS (INC)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), LIttoral ENvironnement et Sociétés (LIENSs), La Rochelle Université (ULR)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne (UCA)-Institut national polytechnique Clermont Auvergne (INP Clermont Auvergne), Université Clermont Auvergne (UCA)-Université Clermont Auvergne (UCA), 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 Paris-Saclay, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: chemistry.chemical_classification, biology, 010405 organic chemistry, [SDV]Life Sciences [q-bio], Aldolase A, Substrate (chemistry), Bond formation, 010402 general chemistry, 01 natural sciences, Pollution, Aldehyde, Combinatorial chemistry, 0104 chemical sciences, Adduct, chemistry, Aldol reaction, Nucleophile, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], biology.protein, Environmental Chemistry, Organic chemistry, [CHIM]Chemical Sciences, Stereoselectivity, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract: International audience; Aldolases are key biocatalysts for stereoselective C–C bond formation allowing access to polyoxygenated chiral units through direct, efficient, and sustainable synthetic processes. The aldol reaction involving unprotected hydroxypyruvate and an aldehyde offers access to valuable polyhydroxy-α-keto acids. However, this undescribed aldolisation is highly challenging, especially regarding stereoselectivity. This reaction was explored using, as biocatalysts, a collection of aldolases selected from biodiversity. Several enzymes that belong to the same pyruvate aldolase Pfam family (PF03328) were found to produce the desired hexulosonic acids from hydroxypyruvate and D-glyceraldehyde with complementary stereoselectivities. One of them was selected for the proof of concept as a biocatalytic tool to prepare five (3S,4S) aldol adducts through an eco-friendly process.
Published: 2017

20. Large α-aminonitrilase activity screening of nitrilase superfamily members: Access to conversion and enantiospecificity by LC–MS

Author: David Vallenet, Virginie Pellouin, Jean-Louis Petit, Jean Weissenbach, Marcel Salanoubat, Franck Bordier, Carine Vergne-Vaxelaire, Aurélie Fossey, Johanna Rohault, Adrien Debard, Véronique de Berardinis, Alain Perret, Ekaterina Darii, Sabine Tricot, Mark Stam, Aline Mariage, Anne Zaparucha, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), 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 Paris-Saclay-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 Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), CH Valenciennes, Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), 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 d'Évry-Val-d'Essonne (UEVE), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)
Subjects: chemistry.chemical_classification, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, biology, Chemistry, Stereochemistry, Process Chemistry and Technology, Burkholderia xenovorans, Substrate (chemistry), Bioengineering, SUPERFAMILY, [CHIM.CATA]Chemical Sciences/Catalysis, biology.organism_classification, Biochemistry, Nitrilase, Catalysis, Hydrolysis, Enzyme, Liquid chromatography–mass spectrometry, [CHIM]Chemical Sciences, Enantiomeric excess, ComputingMilieux_MISCELLANEOUS
Abstract: A high-throughput screening for the identification of nitrilases demonstrating activity towards alpha-aminonitriles is reported. A LC–MS assay giving access to both conversion and enantiospecificity was developed. 588 candidate enzymes were screened as cell lysates against six alpha-aminonitriles in 96-well microplates. The candidate enzymes were selected following two criteria, their sequence identity with a set of known nitrilases or their phylogenetic position among the nitrilase superfamily. Five enzymes were identified and found to hydrolyse alpha-aminonitrile into the corresponding alpha-aminoacid. The substrate range was found to be very narrow as only two different alpha-aminonitriles, 2-aminovaleronitrile and 2-amino-2-phenylacetonitrile, were found to be substrates. The biocatalytic capabilities of three enzymes were further investigated and the best result was obtained with an enzyme from Burkholderia xenovorans catalysing the enantiospecific hydrolysis of 2-aminovaleronitrile into (S)-norvaline with excellent conversion and enantiomeric excess.
Published: 2014

21. Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis

Author: Aline Mariage, Agnès Pinet-Turpault, Antoine Danchin, Ekaterina Darii, Véronique de Berardinis, Claudine Médigue, Carine Vergne-Vaxelaire, Anne Zaparucha, François Artiguenave, Clémence Brewee, David Vallenet, Thomas Bessonnet, Jean-Louis Petit, Pascal Bazire, Jean Weissenbach, Marcel Salanoubat, Virginie Pellouin, Marielle Besnard-Gonnet, Alain Perret, Adrien Debard, Karine Bastard, Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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), Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), This work was supported by Commissariat à l’énergie atomique et aux énergies alternatives (CEA), the CNRS and the University of Evry Val d’Essonne, Savelli, Bruno, 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 Paris-Saclay, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP]-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)
Subjects: 0301 basic medicine, Genetics, chemistry.chemical_classification, Acinetobacter, 030106 microbiology, Cell Biology, Biology, Genome, Methionine biosynthesis, Evolution, Molecular, 03 medical and health sciences, chemistry.chemical_compound, Metabolic pathway, Enzyme, Methionine, Biosynthesis, chemistry, Acetyltransferases, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Homologous chromosome, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Parallel evolution, Molecular Biology, Function (biology)
Abstract: MetA and MetX are phylogenetically unrelated families of acyl-L-homoserine transferases. Experimental assignation of function and structural modeling of these families correct widespread misannotation, reveal convergence of function and uncover new functions in a subclass of MetX. Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.
Published: 2016

22. Possible conformational change within the desolvated and cationized sBBI/trypsin non-covalent complex during the collision-induced dissociation process

Author: Sandra Alves, Jean-Claude Tabet, Ekaterina Darii, Ivo Gut, Gunalini Saravanamuthu, and Carlos Afonso
Subjects: 0303 health sciences, Conformational change, Chromatography, Collision-induced dissociation, Chemistry, Metal ions in aqueous solution, Electrospray ionization, 010401 analytical chemistry, Organic Chemistry, Trypsin, 01 natural sciences, Dissociation (chemistry), 0104 chemical sciences, Analytical Chemistry, 03 medical and health sciences, Crystallography, Protein structure, medicine, Molecule, Spectroscopy, 030304 developmental biology, medicine.drug
Abstract: Electrospray ionization mass spectrometry (ESI-MS) has become an analytical technique widely used for the investigation of non-covalent protein-protein and protein-ligand complexes due to the soft desolvation conditions that preserve the stoichiometry of the interacting partners. Dissociation studies of solvated or desolvated complexes (in the source and in the collision cell, respectively) allow access to information on protein conformation and localization of the metal ions involved in protein structure stabilization and biological activity. The complex of bovine trypsin and small soybean Bowman-Birk inhibitor (sBBI) was studied by ESI-MS to determine changes occurring within the complex during its transfer from droplets to the gas phase independently of the ion polarity. Under collision-induced dissociation (CID) conditions, unexpected binding of the Ca(2+) ion (cofactor of native trypsin) to the inhibitor molecule was observed within the desolvated sBBI/trypsin/Ca(2+) complex (with a 1:1:1 stoichiometry). This formal gas-phase migration of the calcium ion from trypsin to the inhibitor may be related to conformational rearrangements in the solvent-free and likely collapsed complex. However, under conditions leading to the increase in complex charge state, the appearance of the cationized trypsin molecule was detected during complex dissociation, thus reflecting different pathways of the evolution of complex conformation.
Published: 2011

23. Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist’s Toolbox

Author: Alain Perret, Marielle Besnard-Gonnet, Véronique de Berardinis, Marine Debacker, Virginie Pellouin, Jean-Louis Petit, Marcel Salanoubat, Adrien Debard, Marielle Lemaire, Aline Mariage, Virgil Hélaine, Ekaterina Darii, Carlos A. F. Fernandes, Martine Sancelme, Christine Guérard-Hélaine, Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Clermont-Ferrand (ICCF), SIGMA Clermont (SIGMA Clermont)-Institut de Chimie du CNRS (INC)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Sigma CLERMONT (Sigma CLERMONT), SIGMA Clermont (SIGMA Clermont)-Centre National de la Recherche Scientifique (CNRS)-Université Blaise Pascal - Clermont-Ferrand 2 (UBP), 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 Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-SIGMA Clermont (SIGMA Clermont)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV.BIO]Life Sciences [q-bio]/Biotechnology, biocatalysis, [SDV]Life Sciences [q-bio], enzymes, Dihydroxyacetone, 01 natural sciences, Catalysis, Inorganic Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Aldol reaction, Sequence comparison, biocatalysis, C[BOND]C coupling, enzymes, gene expression, mass spectrometry, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Physical and Theoretical Chemistry, 030304 developmental biology, mass spectrometry, chemistry.chemical_classification, 0303 health sciences, Multiple sequence alignment, biology, 010405 organic chemistry, Organic Chemistry, Aldolase A, [CHIM.CATA]Chemical Sciences/Catalysis, 0104 chemical sciences, Enzyme, chemistry, Biochemistry, Biocatalysis, biology.protein, C[BOND]C coupling, gene expression, Genome mining
Abstract: International audience; Stereoselective carboligating enzymes were discovered by a genome mining approach to extend the biocatalysis toolbox. Seven hundred enzymes were selected by sequence comparison from diverse prokaryotic species as representatives of the aldolase (FSA) family diversity. The aldol reaction tested involved dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate. The hexose-6-phosphate formation was monitored by mass spectrometry. Eighteen enzymes annotated either as transaldolases or aldolases were found to exhibit a DHA aldolase activity. Remarkably, six of them proven as aldolases, and not transaldolases, shared very limited similarities with those currently described. Multiple sequence alignment performed on all enzymes revealed a Tyr in the new DHA aldolases as found in FSAcoli instead of a Phe usually found in transaldolases. Four of these DHA aldolases were biochemically characterised in comparison with FSAcoli. In particular, an aldolase from Listeria monocytogenes exhibited interesting catalytic properties.
Published: 2015

24. Structural studies of the sBBI/trypsin non‐covalent complex using covalent modification and mass spectrometry: Structural studies of the sBBI/trypsin non‐covalent complex

Author: Guanalini Saravanamuthu, Ekaterina Darii, Jean-Claude Tabet, Ivo Gut, Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centro Nacional de Análisi Genómico (CNAG), Centro Nacional de Análisis Genómico, Chimie Structurale Organique et Biologique (CSOB), Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Chromatography, Chemistry, Stereochemistry, 010401 analytical chemistry, Organic Chemistry, Mass spectrometry, Tandem mass spectrometry, Trypsin, 01 natural sciences, Dissociation (chemistry), 0104 chemical sciences, Analytical Chemistry, Ion, 03 medical and health sciences, Covalent bond, Desorption, medicine, Molecule, [CHIM]Chemical Sciences, Spectroscopy, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, medicine.drug
Abstract: RATIONALE The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes. METHODS Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. RESULTS Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag). CONCLUSIONS The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi/yj ion series depending on the positions of the tags. Copyright © 2014 John Wiley & Sons, Ltd.
Published: 2014

25. Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry

Author: Ekaterina, Darii, Guanalini, Saravanamuthu, Ivo G, Gut, and Jean-Claude, Tabet
Subjects: Models, Molecular, Proline, Tandem Mass Spectrometry, Molecular Sequence Data, Trypsin, Amino Acid Sequence, Hydrogen-Ion Concentration, Peptides, Trypsin Inhibitor, Bowman-Birk Soybean
Abstract: The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes.Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis.Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag).The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi /yj ion series depending on the positions of the tags.
Published: 2013

26. Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

Author: Jörg Tost, Andrei Alexandrovich Stomakhin, Nelly Papin, Diane Lebeau, Ekaterina Dementieva, Ekaterina Darii, Sascha Sauer, Alla Rubina, Alexander S. Zasedatelev, E. N. Savvateeva, Ivo Gut, and Alexander A. Makarov
Subjects: Immunoassay, Proteomics, Chromatography, Resolution (mass spectrometry), Protein mass spectrometry, Chemistry, Quantitative proteomics, Protein Array Analysis, Proteins, Bioengineering, Hydrogels, General Medicine, Tandem mass tag, Isotope-coded affinity tag, Label-free quantification, Isobaric labeling, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Time-of-flight mass spectrometry, Molecular Biology, Biotechnology
Abstract: Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to the resolution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Arrays and MALDI Mass Spectrometry.
Published: 2008

27. Improvements of TArgeted multiplex mass spectrometry IMaging

Author: Benoit Terris, Mohamed Abarbri, Ivo Gut, Elsa Anselmi, Anne Audebourg, Gwendoline Thiery, Jean-Claude Tabet, and Ekaterina Darii
Subjects: biology, Chemistry, Tissue Array Analysis, Mass spectrometry, Proteomics, Biochemistry, Molecular biology, Primary and secondary antibodies, Immunohistochemistry, Mass spectrometry imaging, Langerhans Cells, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Imaging technology, biology.protein, Humans, Multiplex, Molecular Biology, Pancreas, Biomarkers, Biomedical engineering
Abstract: MALDI mass spectrometers have become popular tools for imaging histological sections. Currently this technology is primarily used for imaging naturally occurring molecules. Here we report on the improvement of TArgeted multiplex MS IMaging (TAMSIM) technology. For TAMSIM we attach photocleavable mass tags to antibodies. Staining histological sections is done analogously to standard immunohistochemical procedures with chemiluminescent or fluorescent detection with the sole difference that multiple antibodies each with a distinct mass tag are used in a single reaction. Mass tags are released from their respective antibodies by a laser pulse at 355 nm without added matrix. After scanning, MS images are created for each tag mass. The enhancements of TAMSIM presented here relate to four elements, the use of an improved generation of tags, their conjugation directly to primary antibodies, the comparison of fresh frozen sections with paraffin embedded ones for the TAMSIM imaging technology and finally, the increase of multiplex detection. Sections of healthy human pancreatic tissue were imaged to visualize different specific biomarkers (synaptophysin, chromogranin, insulin, calcitonin, somatostatin) in neuroendocrine cells of Langerhans islets. The aim was to localize these biomarkers on the tissue sections simultaneously.
Published: 2008

28. Microbial Degradation of a Recalcitrant Pesticide: Chlordecone

Author: Denis Le Paslier, Gabor Gyapay, Valérie Barbe, Thomas Brüls, Aurélie Fossey, Loïc Couturat, Georges N. Cohen, Edgardo Ugarte, Stéphanie Fouteau, Emilie Pateau, Agnès Barbance, Nuria Fonknechten, Sébastien Chaussonnerie, Pierre-Loïc Saaidi, Marion Chevallier, Jean Weissenbach, Delphine Muselet, Institut de Génomique d'Evry (IG), Institut de Biologie François JACOB (JACOB), 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 Paris-Saclay, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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 Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Support was provided by the INRA AIP Demichlord part of PNAC I, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), the CNRS and the University of Evry. MC work was funded by CEA., The authors would like to thank Philippe Bertin, Yves-Marie Cabidoche, Jean-Marie Côme, Stéphane Frenette, Didier Lièvremont, Fabrice Martin-Laurent and Laurent Quillet for providing soils, sediments and wastewater samples. They also thank Anne Zaparucha for helpful discussions, Ekaterina Darii for LC-MS analyses and Laurence Marie for technical skills. This article is dedicated to the memory of Dr. Yves-Marie Cabidoche., Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Microbiology (medical), lcsh:QR1-502, Environmental pollution, 010501 environmental sciences, 01 natural sciences, Microbiology, anaerobic biodegradation, lcsh:Microbiology, Citrobacter amalonaticus, 03 medical and health sciences, chemistry.chemical_compound, Citrobacter, Biotransformation, analytical chemistry, Microbial biodegradation, 0105 earth and related environmental sciences, Original Research, metagenomics, chlordecone, biology, Ecology, kepone, pesticides, Pesticide, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 6. Clean water, 3. Good health, 030104 developmental biology, chemistry, 13. Climate action, Environmental chemistry, organochlorine, Kepone, GC-MS, Microcosm
Abstract: International audience; Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 μg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.
Published: 2016

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28 results on '"Ekaterina Darii"'

1. Simplified in Vitro and in Vivo Bioaccess to Prenylated Compounds

2. Exploring natural biodiversity to expand access to microbial terpene synthesis

3. Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations

4. Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes

5. First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures

6. Simplified in Vitro and in Vivo Bioaccess to Prenylated Compounds

7. 2-Deoxyribose-5-phosphate aldolase, a remarkably tolerant aldolase towards nucleophile substrates

8. Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations

9. Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments

10. Exploring natural biodiversity to expand access to microbial terpene synthesis

11. De novo structure determination of 3-((3-aminopropyl)amino)- 4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1

12. Characterization of l-Carnitine Metabolism in Sinorhizobium meliloti

13. Asymmetric reductive amination by a wild-type amine dehydrogenase from the thermophilic bacteria Petrotoga mobilis

14. Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites

15. Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1- phosphate Aldolases

16. Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

17. Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution

18. Meaning and consequence of the coexistence of competitive hydrogen bond/salt forms on the dissociation orientation of non-covalent complexes

19. Expanding the reaction space of aldolases using hydroxypyruvate as a nucleophilic substrate

20. Large α-aminonitrilase activity screening of nitrilase superfamily members: Access to conversion and enantiospecificity by LC–MS

21. Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis

22. Possible conformational change within the desolvated and cationized sBBI/trypsin non-covalent complex during the collision-induced dissociation process

23. Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist’s Toolbox

24. Structural studies of the sBBI/trypsin non‐covalent complex using covalent modification and mass spectrometry: Structural studies of the sBBI/trypsin non‐covalent complex

25. Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry

26. Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

27. Improvements of TArgeted multiplex mass spectrometry IMaging

28. Microbial Degradation of a Recalcitrant Pesticide: Chlordecone

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