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Cantu D.C.,Iowa State University | Ardevol A.,Computer Simulation and Modeling Laboratory | Ardevol A.,ETH Zurich | Rovira C.,University of Barcelona | And 2 more authors.
Chemistry - A European Journal | Year: 2014

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzymeA moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone. The mechanics of a hotdog-fold thioesterase: The reaction of a human thioesterase was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state (indicated as TS on the free-energy surface reconstructed from the metadynamics simulation). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Saade M.,Institute Biologia Molecular Of Barcelona | Gutierrez-Vallejo I.,Institute Biologia Molecular Of Barcelona | LeDreau G.,Institute Biologia Molecular Of Barcelona | Rabadan M.A.,Institute Biologia Molecular Of Barcelona | And 3 more authors.
Cell Reports | Year: 2013

The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis, and these regulatory processes are subverted in tumor formation. Here, we developed markers that provided the single-cell resolution necessary to quantify the three modes of division taking place in the developing nervous system invivo: self-expanding, PP; self-replacing, PN; and self-consuming, NN. Using these markers and a mathematical model that predicts the dynamics of motor neuron progenitor division, we identify a role for the morphogen Sonic hedgehog in the maintenance of stem cell identity in the developing spinal cord. Moreover, our study provides insight into the process linking lineage commitment to neurogenesis with changes in cell-cycle parameters. As a result, we propose a challenging model in which the external Sonic hedgehog signal dictates stem cell identity, reflected in the consequent readjustment of cell-cycle parameters Source


Unno M.,Tohoku University | Unno M.,Ibaraki University | Unno M.,RIKEN | Ardevol A.,Computer Simulation and Modeling Laboratory | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe3+-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe3+-biliverdin-bound and the biliverdinbound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe3+-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at theHmuOheme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe3+-biliverdin and biliverdin complexes has been determined at 1.70Å resolution, implying a possible route for iron exit. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Thompson A.J.,University of York | Dabin J.,University of York | Iglesias-Fernandez J.,Computer Simulation and Modeling Laboratory | Iglesias-Fernandez J.,Institute Of Quimica Tearica I Computacional Iqtcub | And 17 more authors.
Angewandte Chemie - International Edition | Year: 2012

Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Biarnes X.,Computer Simulation and Modeling Laboratory | Biarnes X.,Ramon Llull University | Ardevol A.,Institute Of Quimica Teorica I Computacional Of La Ub | Ardevol A.,Computer Simulation and Modeling Laboratory | And 6 more authors.
Journal of the American Chemical Society | Year: 2011

Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β- glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a 1,4B/1S3 conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction (1,4B/1S 3 -4E/4H3 - 4C 1) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme. © 2011 American Chemical Society. Source

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