title: Structural characterization and substrate specificity of VpAAT1 protein related to ester biosynthesis in mountain papaya fruit.
abstract: The aroma in fruits is an important attribute of quality that influences consumer's acceptance. This attribute is a complex character determined by a set of low molecular weight volatile compounds. In mountain papaya fruit (Vasconcellea pubescens) the aroma is determined mainly by esters, which are produced through an esterification reaction catalyzed by the enzyme alcohol acyltransferase (AAT) that utilizes alcohols and acyl-CoAs as substrates. In order to understand the molecular mechanism involved in the production of esters in this fruit, an AAT gene which has been previously cloned and characterized from mountain papaya (VpAAT1) was expressed in yeasts, and the highest enzyme activity of the recombinant protein was obtained when the enzyme was tested for its ability to produce benzyl acetate. On the other hand, to gain insight the mechanism of action at the molecular level, a structural model for VpAAT1 protein was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The VpAAT1 structure consists of two domains connected by a large crossover loop, with a solvent channel in the center of the structure formed between the two domains. Residues H166 and D170, important for catalytic action, displayed their side chains towards the central cavity of the channel allowing their interaction with the substrates. The conformational interaction between the protein and several ligands was explored by molecular docking simulations, and the predictions obtained were tested through kinetic analysis. Kinetic results showed that the lowest K(M) values were obtained for acetyl-CoA and benzyl alcohol. In addition, the most favorable predicted substrate orientation was observed for benzyl alcohol and acetyl CoA, showing a perfect coincidence between kinetic studies and molecular docking analysis.
authors.affiliation: Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile.
authors.forename: Luis
authors.initials: L
authors.lastname: Morales-Quintana
authors.forename: Lida
authors.initials: L
authors.lastname: Fuentes
authors.forename: Carlos
authors.initials: C
authors.lastname: Gaete-Eastman
authors.forename: Raúl
authors.initials: R
authors.lastname: Herrera
authors.forename: María Alejandra
authors.initials: MA
authors.lastname: Moya-León
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D029441:Carica / Q000033:anatomy & histology* / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009812:Odorants; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D004952:Esters; D010940:Plant Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases