title: Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.
abstract: Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.
authors.affiliation: Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca , Casilla 747, Talca, Chile 3465548.
authors.forename: Luis
authors.initials: L
authors.lastname: Morales-Quintana
authors.forename: María Ximena
authors.initials: MX
authors.lastname: Nuñez-Tobar
authors.forename: María Alejandra
authors.initials: MA
authors.lastname: Moya-León
authors.forename: Raúl
authors.initials: R
authors.lastname: Herrera
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry*; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D055162:Biocatalysis; D031241:Caricaceae / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005638:Fruit / Q000737:chemistry / Q000201:enzymology*; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000214:Acyl Coenzyme A; D010940:Plant Proteins; D011994:Recombinant Proteins; C044181:hexanoyl-coenzyme A; D000217:Acyltransferases