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8 results on '"substrate distortion"'

1. Substrate binding mode and catalytic mechanism of human heparan sulfate D-glucuronyl C5 epimerase.

Author

Debarnot, Claire, Monneau, Yoan R., Roig-Zamboni, Véronique, Delauzun, Vincent, Le Narvor, Christine, Richard, Emeline, Hénault, Jérôme, Goulet, Adeline, Fadel, Firas, Vivès, Romain R., Priem, Bernard, Bonnaffé, David, Lortat-Jacob, Hugues, and Bournea, Yves

Subjects
*HEPARAN sulfate, *EPIMERASES, *EPIMERIZATION, *OLIGOSACCHARIDES, *HEPARIN
Abstract

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, D-glucuronic acid (GlcA), into L-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs. [ABSTRACT FROM AUTHOR]

Published
2019
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2. QM/MM study of catalytic mechanism of Xylanase Cex from Cellulomonas fimi

Author

Liu, Jingli, Zhang, Chunchun, and Xu, Dingguo

Subjects
*XYLANASES, *MOLECULAR conformation, *BIODEGRADATION, *CELLULOSE, *CELLULOMONAS fimi, *NUCLEOPHILIC reactions, *BIOMASS energy, *HYDROLYSIS
Abstract

Abstract: Xylanase Cex from Cellulomonas fimi is a bifunctional enzyme that catalyzes the degradation of both cellulose and xylan. As a result, it might find valuable applications in production of biofuels. In this work, we presented a detailed theoretical investigation of hydrolysis of the xylopentaose molecule catalyzed by Cex, using a hybrid quantum mechanical and molecular mechanical approach. Our results support the experimental observation that the hydrolysis proceeds via the net retention mechanism. More interestingly, our simulations indicate that the xylose unit at −1 binding site should take a boat (B 2,5) conformation as a possible reactive conformer, while the oxo-carbenium ion-like transition states take the combination of B 2,5/O S 2 for glycosylation, and O S 2/O,3 B for deglycosylation. Our molecular dynamics simulations of mutants further suggest that two catalytic residues (E127 and E233) play the vital role in this ring distortion. Indeed, this conformational change is necessary to facilitate the first step of nucleophilic attack by E233 at the anomeric carbon center. [Copyright &y& Elsevier]

Published
2012
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3. Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase

Author

Emeline Richard, Véronique Roig-Zamboni, Hugues Lortat-Jacob, Firas Fadel, Jérôme Hénault, Adeline Goulet, Christine Le Narvor, Yves Bourne, David Bonnaffé, Vincent Delauzun, C. Debarnot, Yoan R. Monneau, Bernard Priem, Romain R. Vivès, 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), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-11-IDEX-0003,IPS,Idex Paris-Saclay(2011), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), LCOM eG2M, Inst Chim Mol & Mat Orsay, LabEx LERMIT,UMR 8182, Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bourne, Yves, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, and Idex Paris-Saclay - - IPS2011 - ANR-11-IDEX-0003 - IDEX - VALID

Subjects
MESH: Heparin, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Oligosaccharides, Isomerase, Uronic acid, catalytic mechanism, Crystallography, X-Ray, 01 natural sciences, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Substrate Specificity, chemistry.chemical_compound, MESH: Structure-Activity Relationship, Glucuronic Acid, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Trisaccharide, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], chemistry.chemical_classification, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Multidisciplinary, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Nuclear magnetic resonance spectroscopy, Heparan sulfate, Biological Sciences, MESH: Carbohydrate Epimerases, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Glucuronic Acid, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: HEK293 Cells, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, heparan sulfate, substrate distortion, Stereochemistry, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Catalysis, Structure-Activity Relationship, 03 medical and health sciences, Humans, Binding site, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, 030304 developmental biology, X-ray crystallography, Binding Sites, MESH: Humans, Heparin, 010405 organic chemistry, Mandelate racemase, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Active site, MESH: Catalysis, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 0104 chemical sciences, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, HEK293 Cells, chemistry, MESH: Binding Sites, C5 epimerization, biology.protein, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: Substrate Specificity, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Carbohydrate Epimerases, MESH: Oligosaccharides
Abstract

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d -glucuronic acid (GlcA), into l -iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS) n substrate or a (IdoA-GlcNS) n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N -sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Published
2019
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4. Two sides of the same coin : How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

Author

Barth, Andreas and Barth, Andreas

Abstract

This review summarises our infrared spectroscopy and density functional theory studies on the mutual interactions between enzymes and their substrates. We investigated phosphoenolpyruvate bound to pyruvate kinase (EC 2.7.1.40, M1 isozyme), ATP bound to the Ca2+-ATPase (SERCA1a), and the aspartylphosphate moiety of the Ca2+-ATPase phosphoenzyme E2P. Conformational changes of the enzymes and distortions of substrate structure are discussed. In all cases, the infrared absorption of the substrate in the enzyme environment could be identified by a combination of reaction-induced difference spectroscopy and isotopic labelling. The experimentally-determined vibrational frequencies were interpreted in structural terms using experimental correlations or modelling of the active site in density functional theory calculations. For none of the three systems, a weakening of the bond that is cleaved in the following enzymatic reaction could be detected in the ground state of the enzyme-substrate complex. However, for the dephosphorylation reaction of the Ca2+-ATPase phosphoenzyme E2P, a high energy intermediate, not detected in experiments, is the reactant state according to density functional theory calculations.

Published
2016
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7. Putative implication of alpha-amylase loop 7 in the mechanism of substrate binding and reaction products release

Author

Tran, V and André-Leroux, Gwenaelle

Subjects
molecular modeling, barley -amylase, loop motion, substrate distortion, robotic calculations, hinge residues
Abstract

Alpha-amylases are widespread endo-enzymes involved in the hydrolysis of internal alpha-(1,4) glycosidic linkages of starch polymers. Molecular modeling of amylose-amylase interactions is a step toward enzymatic mechanism understanding and rational design of new enzymes. From the crystallographic complex of barley alpha-amylase AMY2-acarbose, the static aspects of amylose-amylase docking have been characterized with a model of maltododecaose (DP12) (G. André, A. Buléon, R. Haser, and V. Tran, Biopolymers 1999, Vol. 50, pp. 751-762; G. André and V. Tran, Special Publication no. 246 1999, The Royal Society of Chemistry, H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson, Eds., Cambridge, pp. 165-174). These studies, consistent with the experimental subsite mapping (K. Bak-Jensen, G. André, V. Tran, and B. Svensson, Journal of Biological Chemistry, to be published), propose a propagation scheme for an amylose chain in the active cleft of AMY2. The topographical overview of alpha-amylases identified loop 7 as a conserved segment flanking the active site. Since some crystallographic experiments suspected its high flexibility, its putative motion was explored through a robotic scheme, an alternate route to dynamics simulations that consume CPU time. The present article describes the characteristics of the flexibility of loop 7: location and motion in AMY2. A back-and-forth motion with a large amplitude of more than 0.6 nm was evaluated. This movement could be triggered by two hinge residues. It results in the loop flipping over the active site to enhance the docking of the native helical substrate through specific interactions, it positions the catalytic residues, it distorts the substrate towards its transition state geometry, and finally monitors the release of the products after hydrolysis. The residues involved in the process are now rational mutation points in the hands of molecular biologists.

Published
2004

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