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1. Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides

Author

Mounir Benkoulouche, Akli Ben Imeddourene, Louis-Antoine Barel, Dorian Lefebvre, Mathieu Fanuel, Hélène Rogniaux, David Ropartz, Sophie Barbe, David Guieysse, Laurence A. Mulard, Magali Remaud-Siméon, Claire Moulis, and Isabelle André

Subjects
Medicine, Science
Abstract

Abstract Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active $$\mathrm{\alpha }$$ α -transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.

Published
2021
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2. Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans

Author

David Ropartz, Mathieu Fanuel, Simon Ollivier, Adrien Lissarrague, Mounir Benkoulouche, Laurence A. Mulard, Isabelle André, David Guieysse, Hélène Rogniaux, Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), 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), Chimie des Biomolécules - Chemistry of Biomolecules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), ANR-15-CE07-0019,CarbUniVax,Synergie entre synthèse chimique et ingénierie d'enzymes pour le développement de vaccins glycoconjugués à large spectre(2015), and ANR-18-CE29-0006,ALGAIMS,Analyses de polysaccharides comportant des unités Galf par IRMPD et MS-mobilité ionique(2018)

Subjects
Isomerism, Polysaccharides, Tandem Mass Spectrometry, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Carbohydrates, Oligosaccharides, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Analytical Chemistry
Abstract

International audience; Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerousbiological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD−MS methods for the precise structural characterization of complex glycans.

Published
2022

4. Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts

Author

Sandrine Morel, Etienne Severac, Claire Moulis, David Guieysse, Magali Remaud-Simeon, 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), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, chemistry.chemical_classification, Glycoside Hydrolases, Computer science, Protein design, Oligosaccharides, Green Chemistry Technology, Computational biology, Protein engineering, 010402 general chemistry, 01 natural sciences, Biochemistry, Enzyme structure, 0104 chemical sciences, Analytical Chemistry, 03 medical and health sciences, 030104 developmental biology, Enzyme, chemistry, Glycoside hydrolase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Glycosides, Transglycosylases, Glycoconjugates, Function (biology)
Abstract

International audience; An increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts. The time has now come for researchers to uncover their possibilities and learn how to design and precisely refine their activity to respond more rapidly to the growing demand for well-defined glycosidic structures.

Published
2021

5. A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

Author

Manon Molina, Claire Moulis, David Guieysse, Magali Remaud-Simeon, Nelly Monties, Gianluca Cioci, Sandrine Morel, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, 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), and Université de Toulouse (UT)

Subjects
0301 basic medicine, glucansucrase, carbohydrate structure, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, enzyme catalysis, 03 medical and health sciences, Bacterial Proteins, Protein Domains, Oligosaccharide binding, Leuconostoc citreum, alternan, medicine, Glucansucrase, Glycoside hydrolase, enzyme mechanism, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Glucans, Molecular Biology, alternansucrase, Alanine, Binding Sites, 030102 biochemistry & molecular biology, biology, biosourced, Chemistry, green chemistry, bio-sourced, Glycosyltransferases, sugar-binding pockets, Cell Biology, Processivity, processivity, sugar binding pockets, Alternansucrase, surface binding site, GH70, polymerase, 030104 developmental biology, surface-binding site, polysaccharide, dextran, Enzymology, biology.protein, Leuconostoc, biotechnology
Abstract

International audience; Microbial ?-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) fromLeuconostoc citreumNRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASR?2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln(700)and Tyr(717)) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

Published
2020

6. Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function

Author

Julien Durand, Bernard Henrissat, Nicolas Terrapon, Ao Li, Elisabeth Laville, Vincent Lombard, David Guieysse, Gabrielle Potocki-Véronèse, Jérémy Esque, David Ropartz, Diego P. Morgavi, Laurence Tarquis, 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), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité Mixte de Recherche sur les Herbivores - UMR 1213 (UMRH), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), INSA Toulouse, China Scholarship Council, ANR-16-CE20-0006,OLIGOMET,Châssis métabolique polyvalent pour la production d'oligosaccharides à haute valeur-ajoutée(2016), European Project: 685474,H2020,H2020-LEIT-BIO-2015-1,METAFLUIDICS(2016), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Signal peptide, Genomic Methodologies: Data clustering methods, Glycoside Hydrolases, Phosphorylases, sequence similarity networks, Swine, [SDV]Life Sciences [q-bio], mannosides, Oligosaccharides, medicine.disease_cause, GH130 CAZy family, 03 medical and health sciences, Glycogen phosphorylase, Mice, 0302 clinical medicine, medicine, Animals, Humans, Shigella, Glycoside hydrolase, Microbiome, Amino Acid Sequence, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Bacteria, Base Sequence, General Medicine, Sequence Analysis, DNA, In vitro, Gastrointestinal Microbiome, Enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, chemistry, Cattle, glycoside phosphorylases, gut microbiomes, 030217 neurology & neurosurgery, Intracellular, Research Article
Abstract

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for thein vitrosynthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of theShigellalipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

Published
2020

7. Engineering of Candida antarctica lipase B for poly(ε-caprolactone) synthesis

Author

Magali Remaud-Simeon, Isabelle André, David Guieysse, Stéphane Emond, Frédéric Peruch, Cedric Montanier, Nicolas Chabot, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), 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-Centre National de la Recherche Scientifique (CNRS), 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), ANR-07-CP2D-0015,POLYMER BIO-PATH,BIO-INSPIRED CATALYTIC PLATFORMS AS A SUSTAINABLE ROUTE TO POLYMERS(2007), Institut National de la Recherche Agronomique (INRA)-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), 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), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Lycée Lalande, 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-Centre National de la Recherche Scientifique ( CNRS ), TEAM 1 LCPO, Université de Bordeaux ( UB ) -Institut Polytechnique de Bordeaux-Centre National de la Recherche Scientifique ( CNRS ), and Centre National de la Recherche Scientifique ( CNRS )

Subjects
Materials science, Polymers and Plastics, Stereochemistry, [SDV]Life Sciences [q-bio], General Physics and Astronomy, 010402 general chemistry, 01 natural sciences, Ring-opening polymerization, Catalysis, chemistry.chemical_compound, ring opening polymerization, Materials Chemistry, Saturated mutagenesis, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Candida antarctica lipase B, biology, poly(epsilon-caprolactone), 010405 organic chemistry, fungi, Organic Chemistry, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Active site, Protein engineering, biology.organism_classification, 0104 chemical sciences, [ CHIM.POLY ] Chemical Sciences/Polymers, enzyme engineering, chemistry, biology.protein, Candida antarctica, screening assay, Caprolactone, Lactone
Abstract

Development of novel synthetic routes based on more eco-friendly processes, such as enzyme-based catalysis, are in great demand to produce biopolymers. Among them, enzymatic Ring Opening Polymerization (eROP) of lactones using lipases, such as Candida antarctica lipase B (CalB), offers new opportunities, although they have been mostly explored in wild-type form. In this study, we followed a structure-based engineering strategy to redesign CalB active site in order to improve catalytic efficiency of poly(e-caprolactone) synthesis and size of polymer products. A library of 1410 single mutants was constructed by saturation mutagenesis that targeted 15 amino acid positions of the active site, identified by molecular modelling. Novel High-Throughput Screening (HTS) assays based either on the ring-opening of the lactone or the detection of poly(e-caprolactone) production were developed and used to screen the library. This led to the identification of 8 improved mutants, among which mutant I285R showed nearly 3-fold increase in catalytic efficiency for the e-caprolactone opening, first step of the reaction, and 30% increased poly(e-caprolactone) molar mass compared to parental wild-type CalB enzyme in same reaction conditions.

Published
2017

8. Deciphering an Undecided Enzyme: Investigations of the Structural Determinants Involved in the Linkage Specificity of Alternansucrase

Author

Manon Molina, Claire Moulis, Nelly Monties, Sandra Pizzut-Serin, David Guieysse, Sandrine Morel, Gianluca Cioci, Magali Remaud-Siméon, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and 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)

Subjects
0303 health sciences, crystal structure, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, 010405 organic chemistry, glucansucrase, General Chemistry, +3%29%2Falpha-%281+->+6%29+linkage+specificity+and+alternance%22">alpha-(1 -> 3)/alpha-(1 -> 6) linkage specificity and alternance, 01 natural sciences, Catalysis, 0104 chemical sciences, GH70, 03 medical and health sciences, polymérase, biopolymer, alternan, biopolymère, caractérisation biochimique, 030304 developmental biology, alternansucrase
Abstract

Understanding how polymerases catalyze the synthesis of biopolymers is a timely and important issue in generating controlled structures with well-defined properties. With this objective in mind, here we describe the 2.8 angstrom crystal structure of a truncated version of alternansucrase (ASR) from L. citreum NRRL B-1355. Indeed, ASR is a striking example of alpha-transglucosylase among GH70 glucansucrases, capable of catalyzing high and low molar mass alternan, an alpha-glucan comprising alternating alpha-1,3 and alpha-1,6 linkages in its linear chain. The 3D structure sheds light on the various features involved in enzyme stability. Moreover, docking studies and biochemical characterizations of 17 single mutants and two double mutants enable the key determinants of alpha-1,6 or alpha-1,3 linkage specificity to be located and establish the structural basis of alternance. ASR displays two different acceptor subsites in the prolongation of its subsites -1 and +1. The first one is defined by Trp675, a residue of subsite +2, and orients acceptor binding exclusively toward alpha-1,6 linkage synthesis. The second binding site comprises Asp772 and Trp543, two residues defining the +2' and +3' subsites, respectively, which are critical for alpha-1,3 linkage formation. It is proposed that the interplay between these two acceptor sites controls alternance. These results add to the toolbox of enzymes for the production of tailor-made polysaccharides with controlled structures.

Published
2019

9. Roles of the F-domain in [FeFe] hydrogenase

Author

Isabelle Meynial-Salles, Charles Gauquelin, Isabelle André, Carole Baffert, David Guieysse, Emilien Etienne, Laurence Girbal, Emma Kamionka, Bruno Guigliarelli, Christophe Léger, Vincent Fourmond, Philippe Soucaille, Pierre Richaud, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-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), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-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)-Aix Marseille Université (AMU)-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), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), National French EPR network (RENARD) [IR3443], EU [1944-32670], Provence Alpes Cote d'Azur (PACA) [DEB 09-621], ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Bioénergie et Microalgues (EBM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergie et Microalgues ( EBM ), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) ( BIAM ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -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 ) -Aix Marseille Université ( AMU ) -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 ), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Hydrogenase, Clostridium acetobutylicum, Stereochemistry, [SDV]Life Sciences [q-bio], Mutant, Mutation, Missense, Biophysics, Electron transfer pathway, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, [ CHIM ] Chemical Sciences, H-cluster, 03 medical and health sciences, Electron transfer, [Fe-Fe] hydrogenase, Bacterial Proteins, Protein Domains, Hyda, Ferredoxin, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, biology, accessory domains, Cell Biology, biology.organism_classification, ferredoxin, Enzyme assay, 0104 chemical sciences, 030104 developmental biology, Enzyme, Amino Acid Substitution, chemistry, biology.protein
Abstract

International audience; The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_Delta DA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H(+)/H-2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.

Published
2018

10. Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Author

Juan Cortés, Sophie Puech‐Guenot, Magali Remaud-Simeon, Isabelle André, Pierre Monsan, David Guieysse, Thierry Siméon, Vincent Lafaquière, Sophie Barbe, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Équipe Robotique et InteractionS (LAAS-RIS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Institut des Technologies Avancees du Vivant (ITAV), Toulouse Canceropole campus, Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut National de la Recherche Agronomique (INRA)-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), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-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 Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), and Université de Toulouse (UT)

Subjects
Models, Molecular, Molecular model, Protein Conformation, Stereochemistry, Mutant, Acetates, Molecular Dynamics Simulation, Protein Engineering, 01 natural sciences, Biochemistry, Substrate Specificity, 03 medical and health sciences, [CHIM.GENI]Chemical Sciences/Chemical engineering, Catalytic Domain, enantioselectivity, lipase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, Lipase, Binding site, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Binding Sites, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, molecular modeling, 010405 organic chemistry, Organic Chemistry, Active site, Substrate (chemistry), Stereoisomerism, Chiral resolution, 0104 chemical sciences, Amino acid, enzyme, chemistry, Mutagenesis, Site-Directed, biology.protein, Molecular Medicine, chiral resolution, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM]
Abstract

International audience; [a] ((Dedication, optional)) Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2-substituted racemic acid derivatives which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild-type BCL could be improved by chemical engineering of the substrate molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aiming at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for their enantioselectivity towards (R,S)-2-chloro ethyl 2-bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable 10 times enhanced or reversed enantioselectivity. Analysis of substrate docking and trajectories in the active site was then performed. From this analysis, the construction of 13 double-mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E-value of 178 and a 15 times enhanced specific activity compared to the parental enzyme, thus demonstrating the efficiency of the semi-rational engineering strategy.

Published
2009

11. New efficient lipase from Yarrowia lipolytica for the resolution of 2-bromo-arylacetic acid esters

Author

Georgina Sandoval, Laeticia Faure, David Guieysse, Pierre Monsan, Jean-Marc Nicaud, Alain Marty, Unité mixte de recherche biotechnologies bioprocédés, Institut National de la Recherche Agronomique (INRA)-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), Microbiologie et Génétique Moléculaire (MGM), Institut National de la Recherche Agronomique (INRA)-Institut National Agronomique Paris-Grignon (INA P-G)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Stereochemistry, [SDV]Life Sciences [q-bio], BURKHOLDERIA CEPACIA, Rhizomucor miehei, ENANTIOSELECTIVITY, 010402 general chemistry, 01 natural sciences, Catalysis, Inorganic Chemistry, 03 medical and health sciences, TRANSESTERIFICATION, [SDV.IDA]Life Sciences [q-bio]/Food engineering, RHIZOMUCOR MIEHEL, Organic chemistry, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Physical and Theoretical Chemistry, Lipase, LIP2, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, 010405 organic chemistry, Organic Chemistry, Yarrowia, General Medicine, Transesterification, biology.organism_classification, GENETIC ENGINEERING, YARROWIA LIPOLYTICA, Yeast, 0104 chemical sciences, Enzyme, Burkholderia, chemistry, ETHYL ESTER, biology.protein, LIPASE
Abstract

International audience; A new extracellular lipase (Lip2p) from the yeast Yarrowia lipolytica was used for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates for the pharmaceutical industry. Its efficiency for the transesterification of racemic mixtures with 1-octanol in n-octane was compared with the most efficient lipases described to date, lipases from Burkholderia cepacia and Rhizomucor miehei. Resolution of 2-bromo-p-tolylacetic acid ethyl ester catalyzed by Y. lipolytica lipase showed an enantio preference of 28, almost equal to that obtained with B. cepacia lipase (E = 30). Moreover, Y lipolytica lipase presents a higher catalytic activity and an (S)-enantiopreference, while B. cepacia lipase is (R)-enantiomer selective. The most interesting result is that Y lipolytica lipase has until now been the only enzyme able to catalyze the resolution of 2-bromo-o-tolylacetic acid ethyl ester (E = 27).

Published
2004

12. Towards a novel explanation of Pseudomonas cepacia lipase enantioselectivity via molecular modelling of the enantiomer trajectory into the active site

Author

Pierre Monsan, Vinh Tran, Christophe Salagnad, David Guieysse, and Magali Remaud-Simeon

Subjects
Substrate Interaction, biology, Hydrogen bond, Chemistry, Stereochemistry, Organic Chemistry, Active site, Substrate (chemistry), Catalysis, Inorganic Chemistry, Tetrahedral carbonyl addition compound, biology.protein, Side chain, Physical and Theoretical Chemistry, Lipase, Enantiomer
Abstract

In the transesterification reaction between ( RS )-2-bromophenyl acetic acid ethyl ester and 1-octanol in n -octane, Pseudomonas cepacia lipase enantioselectivity towards the ( R )-isomer is 57. Two strategies are described to investigate the structural basis involved in this enzyme enantioselectivity. Molecular modelling of the tetrahedral intermediate mimicking the transition state enables the identification of two potentially productive substrate-binding modes for each enantiomer. However, the conformations obtained with the faster and slower-reacting enantiomers have equivalent potential energies and most of them possess the hydrogen bonds essential for catalysis. On this basis, it is not possible to distinguish the diastereomeric complexes. The second approach is original and consists in a simple but robust protocol of pseudomolecular dynamics simulations under constraints to map the probable trajectory of the enantiomers in the active site. Enzyme/substrate interaction energy is always found to be lower for the faster-reacting enantiomer, which satisfactorily corroborates the experimental results. Energy differences are attributed to specific interactions of these substrates with a network of hydrophobic residues lining the access path. Furthermore, mechanistic details suggest that the pivoting side chains of the hydrophobic residues act in a concerted step–tooth gear motion whose basic role is to select and guide the substrates towards the active site. With this type of lipase, such dynamic features could be the key explanation of this as yet unexplored enantiorecognition. For the slower-reacting enantiomer, it appears that the concerted motion of the side chains is perturbed when the substrate passes through a bottleneck formed by Val266 and Leu17. The enantioselectivity of mutant Val266Leu with a more bulky side chain at this position supports our assumption: by narrowing the bottleneck, the enantioselectivity was considerably enhanced as much as up to 200.

Published
2003

13. Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia

Author

Wouter F. Slotema, Adrie J. J. Straathof, David Guieysse, Alain Marty, and Georgina Sandoval

Subjects
Oleamide, Butanols, Triacylglycerol lipase, Oleic Acids, Pilot Projects, Bioengineering, Applied Microbiology and Biotechnology, Catalysis, Fungal Proteins, Ammonia, chemistry.chemical_compound, Pentanols, Amide, Organic chemistry, Computer Simulation, Lipase, Plug flow reactor model, biology, Chemistry, Enzymes, Immobilized, biology.organism_classification, Amides, United States, Europe, Oleic acid, Models, Chemical, Solvents, biology.protein, Candida antarctica, Oleic Acid, Biotechnology
Abstract

An economically pertinent process for the lipase-catalyzed synthesis of amides was developed. A continuous plug flow reactor was used. The model reaction was the production of oleamide, a lubricant and anti-slip agent, via direct Candida antarctica lipase B-catalyzed amidation of oleic acid with ammonia. Of all solvents tested, 2-methyl-2-butanol was found to respond optimally to the demands formulated in our specifications. A continuous conversion of oleic acid into oleamide of 85% was obtained. A productivity of 4.5 tons oleamide per kg of enzyme per year was calculated, indicating a contribution of enzyme to the product price of only 4%. The volumetric productivity, 100 g. L(-1). h(-1), is 4 to 100 times higher than in literature procedures. A simple crystallization procedure leads to 99% purity.

Published
2003

14. Lipase-catalyzed enantioselective transesterification toward esters of 2-bromo-tolylacetic acids

Author

Christophe Salagnad, Pierre Monsan, David Guieysse, and Magali Remaud-Simeon

Subjects
chemistry.chemical_classification, biology, Chemistry, Organic Chemistry, Substituent, Enantioselective synthesis, Rhizomucor miehei, Transesterification, biology.organism_classification, Catalysis, Kinetic resolution, Inorganic Chemistry, chemistry.chemical_compound, biology.protein, Organic chemistry, Candida antarctica, Physical and Theoretical Chemistry, Lipase, Alkyl
Abstract

Lipases from Candida antarctica, Pseudomonas cepacia and Rhizomucor miehei were tested in the resolution of seven racemic substrates belonging to the (RS)-2-bromo tolyl acetate ester category, but differing either in the position of the methyl substituent on the acyl part of the aromatic ring, or in the structure of the alkyl group. Lipase-catalyzed kinetic resolution via transesterification reaction between the ester and octanol in octane revealed that, of the three enzymes tested, P. cepacia lipase is the most efficient for resolution of the various racemates, with R-enantiopreference. In addition, the position of the methyl substituent was found to play a key role in governing the enantioselectivity of the reaction. Using P. cepacia lipase and 2-bromo-m/p-tolyl- or 2-bromophenylacetic acid esters E-values of >50 were measured, whereas with the ortho derivatives, E-values dramatically decreased to

Published
2003

15. Comparison of synthetic dye decolorization by whole cells and a laccase enriched extract from Trametes versicolor DSM11269

Author

Monnat Theerachat, David Guieysse, Magali Remaud-Simeon, Sandrine Morel, and Warawut Chulalaksananukul

Subjects
Laccase, biology, ALIZARIN RED, Amaranth, biology.organism_classification, Applied Microbiology and Biotechnology, Anthraquinone, Remazol Brilliant Blue R, Indigo, chemistry.chemical_compound, chemistry, Indigo carmine, Synthetic dyes, Trametes versicolor, decolorization, white-rot fungus, Botany, Genetics, Agronomy and Crop Science, Molecular Biology, Biotechnology, Trametes versicolor, Nuclear chemistry
Abstract

Trametes versicolor strain DSM 11269 was found to decolorize six out of seven different synthetic dyes when grown on dye-containing agar plates. Using a laccase enzyme extract, enriched from the fungal liquid culture supernatant, the anthraquinone derivative dyes (Alizarin Red S and Remazol Brilliant Blue R) were decolorized in three hours at 50°C by 55 and 70%, respectively. The four azo compounds (Amaranth, Cibacron Brilliant Red 3B-A, Direct Blue 71 and Reactive Black 5), and the indigo molecule (Indigo Carmine), showed a higher resistance to decolorization (

Published
2014

16. Resolution of 2-bromo-o-tolyl-carboxylic acid by transesterification using lipases from Rhizomucor miehei and Pseudomonas cepacia

Author

Christophe Salagnad, Pierre Monsan, David Guieysse, and Magali Remaud-Simeon

Subjects
chemistry.chemical_classification, biology, Chemistry, Carboxylic acid, Organic Chemistry, Enantioselective synthesis, Rhizomucor miehei, Transesterification, biology.organism_classification, Catalysis, Inorganic Chemistry, Acetic acid, chemistry.chemical_compound, biology.protein, Organic chemistry, Racemic mixture, Stereoselectivity, Physical and Theoretical Chemistry, Lipase
Abstract

Several lipases were screened for their ability to catalyze the enantioselective transesterification of 2-bromo-o-tolyl acetic acid. Amongst the preparations tested, the lipases from Rhizomucor miehei and Pseudomonas cepacia were selected. The best enantioselectivity was obtained with Rhizomucor miehei lipase immobilized on polypropylene (E=11.3), which was more stereoselective than the free form. Hydrophobic solvents with log P higher than 2.5 were the most suitable giving the highest E-values. In addition, factors such as the water activity and the reaction temperature had little effect on the resolution of the racemic mixture. The selectivity of the enzymes with respect to the substrate was also only weakly affected by the structure of the leaving alcohol except in the case of the iso-propyl group, which causes high steric hindrance. Operating conditions under reduced pressure were defined to resolve the racemic mixture with immobilized Rhizomucor miehei lipase.

Published
2001

17. Probing Substrate Promiscuity of Amylosucrase from Neisseria polysaccharea

Author

David Guieysse, Magali Remaud-Simeon, David Daudé, Isabelle André, Sandrine Morel, Elise Champion, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), French Ministry of Research, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), and 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)

Subjects
ENZYME, Molecular model, glycodiversification, Stereochemistry, GEOTHERMALIS DSM 11300, substrate promiscuity, [SDV]Life Sciences [q-bio], enzymes, 01 natural sciences, Catalysis, Inorganic Chemistry, transglucosylation, 03 medical and health sciences, Amylosucrase, stomatognathic system, RECOMBINANT AMYLOSUCRASE, Neisseria polysaccharea, CRYSTAL-STRUCTURE, Physical and Theoretical Chemistry, CHEMOENZYMATIC SYNTHESIS, 030304 developmental biology, OLIGOSACCHARIDE SYNTHESIS, chemistry.chemical_classification, SUCROSE ANALOGS, 0303 health sciences, biology, 010405 organic chemistry, DEINOCOCCUS-GEOTHERMALIS, ACTIVE-SITE, Organic Chemistry, Active site, glycosides, biology.organism_classification, PYRANOSE DEHYDROGENASE, Acceptor, 0104 chemical sciences, Enzyme, Biochemistry, chemistry, Docking (molecular), biology.protein, Deinococcus geothermalis
Abstract

The amylosucrase from Neisseria polysaccharea (NpAS) naturally catalyzes the synthesis of a variety of products from sucrose and shows signs of plasticity of its active site. To explore further this promiscuity, the tolerance of amylosucrase towards different donor and acceptor substrates was investigated. The selection of alternate donor substrates was first made on the basis of preliminary molecular modeling studies. From 11 potential donors harboring selective derivatizations that were experimentally evaluated, only p-nitrophenyl-α-D-glucopyranoside was used by the wild-type enzyme, and this underlines the high specificity of the −1 subsite of NpAS for glucosyl donor substrates. The acceptor substrate promiscuity was further explored by screening 20 hydroxylated molecules, including D- and L-monosaccharides as well as polyols. With the exception of one compound, all were successfully glucosylated, and this showcases the tremendous plasticity of the +1 subsite of NpAS, which is responsible for acceptor recognition. The products obtained from the transglucosylation reactions of three selected acceptors were characterized, and they revealed original structures and enzyme enantiopreference, which were more particularly analyzed by in silico docking analyses.

Published
2013

18. Alteration of enzyme activity and enantioselectivity by biomimetic encapsulation in silica particles

Author

Pierre Monsan, Severine Lechevallier, Jeannette Dexpert-Ghys, Magali Remaud-Simeon, Stéphane Emond, David Guieysse, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Centre d'élaboration de matériaux et d'études structurales (CEMES), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), European Union [SANTS], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut de Chimie du CNRS (INC)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), 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), Université de Toulouse (UT), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut de Chimie de Toulouse (ICT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
PROTEINS, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Cylindrotheca fusiformis, Peptide, 02 engineering and technology, Butyrate, 010402 general chemistry, 01 natural sciences, Esterase, Catalysis, Hydrolysis, Biomimetic Materials, Biomimetics, BIOSILICA, Enzyme Stability, SUPPORT, Materials Chemistry, Chemical Precipitation, Amino Acid Sequence, YARROWIA-LIPOLYTICA, Lipase, chemistry.chemical_classification, Diatoms, Chromatography, biology, Chemistry, Metals and Alloys, Esterases, EXPRESSION SYSTEM, General Chemistry, 021001 nanoscience & nanotechnology, Enzymes, Immobilized, Silicon Dioxide, Enzyme assay, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Enzyme Activation, Enzyme, Biochemistry, IMMOBILIZATION, Ceramics and Composites, biology.protein, LIPASE, 0210 nano-technology, Peptides
Abstract

Direct encapsulation of esterase or lipase fused with the silica-precipitating R5 peptide from Cylindrotheca fusiformis in silica particles afforded high yields of active entrapped protein. The hydrolytic activity of both enzymes against p-nitrophenyl butyrate was similarly affected by encapsulation and the enantioselectivity of the esterase was both improved and inverted.

Published
2012

19. Structural Investigation of the Thermostability and Product Specificity of Amylosucrase from the BacteriumDeinococcus geothermalis

Author

Gabrielle Potocki-Véronèse, Sophie Barbe, Samuel Tranier, Sandra Pizzut-Serin, Isabelle André, Lionel Mourey, Magali Remaud-Simeon, Pierre Monsan, David Guieysse, Frédéric Guérin, Valérie Guillet, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France, Center of Region Midi-Pyrenees (CALMIP, Toulouse, France), Center for Computing Resources (CRI) of INSA-Toulouse, Poles de Recherche et d'Enseignement Superieur de I'Universite de Toulouse, Region Midi-Pyrenees, France, Institut National de la Recherche Agronomique (INRA)-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), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Sucrose, Hot Temperature, ALPHA-AMYLASE, [SDV]Life Sciences [q-bio], Crystallography, X-Ray, Disaccharides, 01 natural sciences, Biochemistry, Substrate Specificity, Turanose, chemistry.chemical_compound, Enzyme Stability, Deinococcus, CRYSTAL-STRUCTURE, Thermostability, 0303 health sciences, biology, Chemistry, Enzyme structure, Glucosyltransferases, Protein Structure and Folding, Dimerization, Fructose binding, Stereochemistry, PROTEINS, Fructose, SUCROSE ISOMERASES, 010402 general chemistry, SEQUENCE, 03 medical and health sciences, Amylosucrase, Bacterial Proteins, Isomerism, Protein Structure, Quaternary, NEISSERIA-POLYSACCHAREA, Molecular Biology, 3-DIMENSIONAL STRUCTURE, 030304 developmental biology, Cell Biology, biology.organism_classification, Protein Structure, Tertiary, MOLECULAR-DYNAMICS, FORCE-FIELD, MODEL, 0104 chemical sciences, Glucose, Structural biology, biology.protein, Deinococcus geothermalis
Abstract

Amylosucrases are sucrose-utilizing alpha-transglucosidases that naturally catalyze the synthesis of alpha-glucans, linked exclusively through alpha 1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.

Published
2011

20. ChemInform Abstract: Resolution of 2-Bromo-o-tolyl-carboxylic Acid by Transesterification Using Lipases from Rhizomucor miehei and Pseudomonas cepacia

Author

David Guieysse, Magali Remaud-Simeon, Christophe Salagnad, and Pierre Monsan

Subjects
chemistry.chemical_classification, biology, Chemistry, Carboxylic acid, Enantioselective synthesis, Rhizomucor miehei, General Medicine, Transesterification, biology.organism_classification, Acetic acid, chemistry.chemical_compound, biology.protein, Organic chemistry, Racemic mixture, Stereoselectivity, Lipase
Abstract

Several lipases were screened for their ability to catalyze the enantioselective transesterification of 2-bromo-o-tolyl acetic acid. Amongst the preparations tested, the lipases from Rhizomucor miehei and Pseudomonas cepacia were selected. The best enantioselectivity was obtained with Rhizomucor miehei lipase immobilized on polypropylene (E=11.3), which was more stereoselective than the free form. Hydrophobic solvents with log P higher than 2.5 were the most suitable giving the highest E-values. In addition, factors such as the water activity and the reaction temperature had little effect on the resolution of the racemic mixture. The selectivity of the enzymes with respect to the substrate was also only weakly affected by the structure of the leaving alcohol except in the case of the iso-propyl group, which causes high steric hindrance. Operating conditions under reduced pressure were defined to resolve the racemic mixture with immobilized Rhizomucor miehei lipase.

Published
2010

21. Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

Author

Magali Remaud-Simeon, Pierre Monsan, Isabelle André, Sophie Barbe, David Guieysse, Vincent Lafaquière, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), UMR 5504, Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), Computing center of Region Midi-Pyrenees (CALMIP, Toulouse, France), Center for Computing Resources (CRI) of INSA-Toulouse, Institut des Technologies Avancees do Vivant (ITAV), Toulouse Canceropole campus, 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), and Toulouse Biotechnology Institute (TBI)

Subjects
Models, Molecular, Conformational change, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Time Factors, Stereochemistry, Protein Conformation, Burkholderia cepacia, 01 natural sciences, Biochemistry, Phosphates, lid movement, 03 medical and health sciences, Molecular dynamics, Protein structure, Structural Biology, Organic chemistry, Lipase, Enzyme Inhibitors, Molecular Biology, 030304 developmental biology, 0303 health sciences, conformational transition, biology, 010405 organic chemistry, Chemistry, protein flexibility, molecular dynamic, Substrate (chemistry), Active site, Computational Biology, Proteins, biology.organism_classification, solvent environment, protein activation, 0104 chemical sciences, Burkholderia, Helix, biology.protein, Solvents, sense organs, Burkholderia cepacia lipase
Abstract

International audience; The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so-called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position-restrained MD simulations. Our conclusions indicate that the sole mobility of alpha9 helix side-chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by alpha5 helix movement. The role of selected alpha5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase.

Published
2009

22. A Structure-Controlled Investigation of Lipase Enantioselectivity by a Path-Planning Approach

Author

Sophie Puech‐Guenot, Magali Remaud-Simeon, Thierry Siméon, Vincent Lafaquière, Sophie Barbe, Juan Cortés, Pierre Monsan, Isabelle André, David Guieysse, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-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), Équipe Robotique et InteractionS (LAAS-RIS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-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 Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), and Université Fédérale Toulouse Midi-Pyrénées

Subjects
Models, Molecular, Molecular model, Stereochemistry, In silico, substrate access/exit pathway, Burkholderia cepacia, 01 natural sciences, Biochemistry, Molecular engineering, Substrate Specificity, 03 medical and health sciences, [CHIM.GENI]Chemical Sciences/Chemical engineering, enantioselectivity, [INFO.INFO-RB]Computer Science [cs]/Robotics [cs.RO], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lipase, Amino acid residue, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, Molecular Biology, 030304 developmental biology, 0303 health sciences, enzymatic resolution, biology, Molecular Structure, 010405 organic chemistry, Chemistry, Organic Chemistry, Substrate (chemistry), Computational Biology, Stereoisomerism, Transesterification, biology.organism_classification, 0104 chemical sciences, Burkholderia, biology.protein, Molecular Medicine, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], lipases molecular modeling
Abstract

International audience; A novel approach based on efficient path‐planning algorithms was applied to investigate the influence of substrate access on Burkholderia cepacia lipase enantioselectivity. The system studied was the transesterification of 2‐substituted racemic acid derivatives catalysed by B. cepacia lipase. In silico data provided by this approach showed a fair qualitative agreement with experimental results, and hence the potential of this computational method for fast screening of racemates. In addition, a collision detector algorithm used during the pathway searches enabled the rapid identification of amino acid residues hindering the displacement of substrates along the deep, narrow active‐site pocket of B. cepacia lipase and thus provided valuable information to guide the molecular engineering of lipase enantioselectivity.

Published
2008

23. Small-scale production of Burkholderia cepacia ATCC21808 lipase adapted to high-throughput screening

Author

David Guieysse, Sophie Puech-Guenot, Magali Remaud-Simeon, Laure Landric-Burtin, Vincent Lafaquière, and Pierre Monsan

Subjects
High-throughput screening, Genetic Vectors, Drug Evaluation, Preclinical, Mutagenesis (molecular biology technique), Gene Expression, Burkholderia cepacia, medicine.disease_cause, Biochemistry, Analytical Chemistry, law.invention, Erlenmeyer flask, FLAG-tag, law, medicine, Escherichia coli, Lipase, DNA Primers, Expression vector, biology, Base Sequence, biology.organism_classification, Recombinant Proteins, Burkholderia, Genes, Bacterial, biology.protein, Mutagenesis, Site-Directed, Molecular Medicine, Biotechnology, Plasmids
Abstract

The screening of variant libraries of recombinant Burkholderia cepacia ATCC21808 lipase generated in Escherichia coli is limited by expression difficulties that are mainly due to the formation of inclusion bodies. To circumvent these difficulties and provide an efficient small-scale screening protocol, the gene encoding the lipase from B. cepacia was expressed in various expression vectors. With the pFLAG-ATS-Lip-Hp construct, expression of up to 6807 U/L of culture was possible in Erlenmeyer flasks. The production protocol was miniaturized in 96 deep-well plates, yielding 1300 U/L of lipase in fusion with the FLAG tag. With this protocol, the activity was determined in less than 10 min for a full plate, with a coefficient of variance of about 25%. For validation, 18 mutants constructed by site-directed mutagenesis on position Valine 266 were screened. Nice variations of activity were detected and found to be in agreement with those obtained in Erlenmeyer flask cultures. The protocol enabled the identification of 5 mutants showing enhanced activity toward para-nitrophenyl butyrate. (Journal of Biomolecular Screening 2008;13:72-79)

Published
2008

24. Cover Picture: Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel (ChemBioChem 17/2009)

Author

Sophie Puech‐Guenot, Juan Cortés, Vincent Lafaquière, Thierry Siméon, Pierre Monsan, Isabelle André, Sophie Barbe, David Guieysse, and Magali Remaud-Simeon

Subjects
biology, Chemistry, Stereochemistry, Organic Chemistry, biology.protein, Molecular Medicine, Substrate (chemistry), Cover (algebra), Lipase, Binding site, Molecular Biology, Biochemistry
Published
2009

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