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22 items found (0 items not approved), displaying 11 to 20.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 α-Mannosidases Santiago Alonso-gil , Alexandra Males , Pearl Z. Fernandes , Spencer J. Williams , Gideon J. Davies , Carme Rovira Journal Of The American Chemical Society DOI: 10.1021/jacs.6b11247 Diamond Proposal Number(s): [13587] Show Abstract ▼ Abstract: Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4C1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the −1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 → B2,5 → 1S5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.	Jan 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Contribution of shape and charge to the inhibition of a family GH99 endo -α-1,2-mannanase Marija Petricevic , Lukasz F. Sobala , Pearl Fernandes , Lluís Raich , Andrew James Thompson , Ganeko Bernardo-seisdedos , Oscar Millet , Sha Zhu , Matthieu Sollogoub , Jesús Jimenez-barbero , Carme Rovira , Gideon J. Davies , Spencer J. Williams Journal Of The American Chemical Society DOI: 10.1021/jacs.6b10075 Diamond Proposal Number(s): [9948] Show Abstract ▼ Abstract: Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via an 1,2-anhydrosugar 'epoxide' mechanism that proceeds through a proposed unusual conformational itinerary. Here, we explore how charge and shape contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) inhibitor of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.	Dec 2016
I02-Macromolecular Crystallography	A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism Yi Jin , Marija Petricevic , Alan John , Lluís Raich , Huw Jenkins , Leticia Portela De Souza , Fiona Cuskin , Harry J. Gilbert , Carme Rovira , Ethan D. Goddard-borger , Spencer J. Williams , Gideon J. Davies Acs Central Science DOI: 10.1021/acscentsci.6b00232 Diamond Proposal Number(s): [9948] Show Abstract ▼ Abstract: The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.	Nov 2016
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation Erandi Lira-navarrete , Matilde De Las Rivas , Ismael Compañón , María Carmen Pallarés , Yun Kong , Javier Iglesias-fernández , Gonçalo J. L. Bernardes , Jesús M. Peregrina , Carme Rovira , Pau Bernadó , Pierpaolo Bruscolini , Henrik Clausen , Anabel Lostao , Francisco Corzana , Ramon Hurtado-guerrero Nature Communications 6 DOI: 10.1038/ncomms7937 PMID: 25939779 Diamond Proposal Number(s): [8035, 10121] Open Access Show Abstract ▼ Abstract: Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.	May 2015
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Evidence for a Boat Conformation at the Transition State of GH76 α-1,6-Mannanases-Key Enzymes in Bacterial and Fungal Mannoprotein Metabolism Andrew J. Thompson , Gaetano Speciale , Javier Iglesias-fernández , Zalihe Hakki , Tyson Belz , Alan Cartmell , Richard J. Spears , Emily Chandler , Max J. Temple , Judith Stepper , Harry J. Gilbert , Carme Rovira , Spencer J. Williams , Gideon J. Davies Angewandte Chemie 127 (18), 5468 - 5472 DOI: 10.1002/ange.201410502 Show Abstract ▼ Abstract: α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an OS2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through OS2 and B2,5≠ conformations, information that should inspire the development of new antifungal agents.	Apr 2015
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism Andrew J. Thompson , Gaetano Speciale , Javier Iglesias-fernández , Zalihe Hakki , Tyson Belz , Alan Cartmell , Richard J. Spears , Emily Chandler , Max J. Temple , Judith Stepper , Harry J. Gilbert , Carme Rovira , Spencer J. Williams , Gideon J. Davies Angewandte Chemie International Edition DOI: 10.1002/anie.201410502 PMID: 25772148 Diamond Proposal Number(s): [7864, 9948] Show Abstract ▼ Abstract: carbohydrates;computational chemistry;conformational analysis;enzymatic mechanisms;glycosidase inhibitors	Mar 2015
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2 Erandi Lira , Javier Iglesias-fernandez , Wesley F. Zandberg , Ismael Compañón , Yun Kong , Francisco Corzana , B. Mario Pinto , Henrik Clausen , Jesús M. Peregrina , David J. Vocadlo , Carme Rovira , Ramon Hurtado-guerrero Angewandte Chemie International Edition 53 (31), 8206 - 8210 DOI: 10.1002/anie.201402781 PMID: 24954443 Diamond Proposal Number(s): [8035] Show Abstract ▼ Abstract: The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.	Jul 2014
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide Erandi Lira , Javier Iglesias-fernández , Wesley F. Zandberg , Ismael Compañón , Yun Kong , Francisco Corzana , B. Mario Pinto , Henrik Clausen , Jesús M. Peregrina , David J. Vocadlo , Carme Rovira , Ramon Hurtado-guerrero Angewandte Chemie 126, 8345 - 8349 DOI: 10.1002/ange.201402781 Diamond Proposal Number(s): [8035] Show Abstract ▼ Abstract: The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bibi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SNi-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.	Jul 2014
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Combined Inhibitor Free-Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate Rohan J. Williams , Javier Iglesias-fernández , Judith Stepper , Adam Jackson , Andrew Thompson , Elisabeth C. Lowe , John White , Harry J. Gilbert , Carme Rovira , Gideon J. Davies , Spencer J. Williams Angewandte Chemie International Edition 53 (4), 1087 - 1091 DOI: 10.1002/anie.201308334 PMID: 24339341 Diamond Proposal Number(s): [7864] Show Abstract ▼ Abstract: Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.	Jan 2014
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The Reaction Coordinate of a Bacterial GH47 α-Mannosidase: A Combined Quantum Mechanical and Structural Approach Andrew Thompson , Jerome Dabin , Javier Iglesias-fernández , Albert Ardèvol , Zoran Dinev , Spencer J. Williams , Omprakash Bande , Aloysius Siriwardena , Carl Moreland , Ting-chou Hu , David K. Smith , Harry J. Gilbert , Carme Rovira , Gideon J. Davies Angewandte Chemie International Edition 51 (44), 10997 - 11001 DOI: 10.1002/anie.201207917 Show Abstract ▼ Abstract: Mannosidases are glycoside hydrolases that face special stereoelectronic challenges in effecting the hydrolysis of the glycosidic bond. In their Communication on page?10997?ff., C. Rovira, G.?J. Davies, and co-workers use QM/MM calculations, supported by X-ray structures of the enzyme with ligands mimicking the substrate, transition state, and product, to show that the free-energy landscape of an isolated alpha-mannoside is shaped on-enzyme into a single conformational itinerary along the reaction coordinate.	Oct 2012

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