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Gotthard G.,Aix - Marseille University | Hiblot J.,Aix - Marseille University | Gonzalez D.,Aix - Marseille University | Chabriere E.,Aix - Marseille University | And 2 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2013

Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-β-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organo phosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Å resolution of OPHC2 are presented. © 2013 International Union of Crystallography. All rights reserved. Source

Masson P.,Institute Of Recherches Biomedicales Des Armees Crssa | Masson P.,CNRS Institute of Pharmacology and Structural Biology | Masson P.,University of Nebraska Medical Center
Biochemistry (Moscow) | Year: 2012

Cholinesterases (ChEs) display a hysteretic behavior with certain substrates and inhibitors. Kinetic cooperativity in hysteresis of ChE-catalyzed reactions is characterized by a lag or burst phase in the approach to steady state. With some sub- strates damped oscillations are shown to superimpose on hysteretic lags. These time dependent peculiarities are observed for both butyrylcholinesterase and acetylcholinesterase from different sources. Hysteresis in ChE-catalyzed reactions can be inter- preted in terms of slow transitions between two enzyme conformers E and E'. Substrate can bind to E and/or E', both Michaelian complexes ES and ?'S can be catalytically competent, or only one of them can make products. The formal reac- tion pathway depends on both the chemical structure of the substrate and the type of enzyme. In particular, damped oscilla- tions develop when substrate exists in different, slowly interconvertible, conformational, and/or micellar forms, of which only the minor form is capable of binding and reacting with the enzyme. Biphasic pseudo-first-order progressive inhibition of ChEs by certain carbamates and organophosphates also fits with a slow equilibrium between two reactive enzyme forms. Hysteresis can be modulated by medium parameters (pH, chaotropic and kosmotropic salts, organic solvents, temperature, osmotic pres- sure, and hydrostatic pressure). These studies showed that water structure plays a role in hysteretic behavior of ChEs. Attempts to provide a molecular mechanism for ChE hysteresis from mutagenesis studies or crystallographic studies failed so far. In fact, several lines of evidence suggest that hysteresis is controlled by the conformation of His438, a key residue in the catalytic triad of cholinesterases. Induction time may depend on the probability of His438 to adopt the operative conformation in the cat- alytic triad. The functional significance of ChE hysteresis is puzzling. However, the accepted view that proteins are in equilib- rium between preexisting functional and non-functional conformers, and that binding of a ligand to the functional form shifts equilibrium towards the functional conformation, suggests that slow equilibrium between two conformational states of these enzymes may have a regulatory function in damping out the response to certain ligands and irreversible inhibitors. This is par- ticularly true for immobilized (membrane bound) enzymes where the local substrate and/or inhibitor concentrations depend on influx in crowded organellar systems, e.g. cholinergic synaptic clefts. Therefore, physiological or toxicological relevance of the hysteretic behavior and damped oscillations in ChE-catalyzed reactions and inhibition cannot be ruled out. © Pleiades Publishing, Ltd., 2012. Source

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