ORIGINAL PAPER Computational study enlightens the structural role of the alcohol acyltransferase DFGWG motif Luis Morales-Quintana 1 & María Alejandra Moya-León 1 & Raúl Herrera 1 Received: 7 May 2015 /Accepted: 13 July 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Alcohol acyltransferases (AAT) catalyze the esteri- fication reaction of alcohols and acyl-CoA into esters in fruits and flowers. Despite the high divergence between AAT en- zymes, two important and conserved motifs are shared: the catalytic HxxxD motif, and the DFGWG motif. The latter is proposed to play a structural role; however, its function re- mains unclear. The DFGWG motif is located in loop 21 and stabilized by a hydrogen bond between residues Y52 and D381. Also, this motif is distant from the HxxxD motif, and most probably without a direct role in the substrate interaction. To evaluate the role of the DFGWG motif, in silico analysis was performed in the VpAAT1 protein. Three mutants (Y52F, D381A and D381E) were evaluated. Major changes (size and shape) in the solvent channels were found, although no differ- ences were revealed in the entire 3D structure. Molecular dy- namics simulations and docking studies described unfavor- able energies for interaction of the mutant proteins with dif- ferent substrates, as well as unfavored ligand orientations in the solvent channel. Additionally, we examined the contribu- tion of different energetic parameters to the total free energy of protein–ligand complexes by the MM-GBSA method. The complexes differed mainly in their van der Waals contribu- tions and have unfavorable electrostatic interactions. VpAAT1, Y52F and D381A mutants showed a dramatic re- duction in the binding capacity to several substrates, which is related to differences in electrostatic potential on the protein surfaces, suggesting that D381 from the DFGWG motif and residue Y52 play a crucial role in maintenance of the adequate solvent channel structure required for catalysis. Keywords Alcohol acyltransferase . Ester biosynthesis . In silico site-directed mutagenesis . Molecular dynamics simulations . MM-GBSA . Vasconcellea pubescens Introduction The contribution of volatile substances (aroma and flavor) is very important for fruit quality and consumer preference in mountain papaya [1]. As described previously, different alco- hols and a large variety of esters have been reported in ripe fruits [1]. In this sense, esters are likely to be the key contrib- utors to their unique aroma. Volatile esters are catalyzed by alcohol acyltransferases (AAT; EC 2.3.1.84) in an esterification reaction between acyl-CoAs and alcohols [2, 3]. Vasconcellea pubescens alcohol acyltransferase ( VpAAT1) —a member of the BAHD superfamily—was isolated and characterized from ripe mountain papaya fruit [4, 5]. VpAAT1 belongs to subfamily III [4], where other AATs that take part in the synthesis of volatile compounds during fruits ripening such as melon [6] and apple [7–9] are grouped. A low sequence similarity can be observed for these proteins; nevertheless, similar acyltransferase function and the pres- ence of two highly conserved motifs are found [10]. How- ever, phylogenetically closely related enzymes, especially those responsible for volatile ester formation, can share up to 90 % identity. In fact, 55–68 % sequence identity is reported for AATs clustered in subgroup III [4]. Electronic supplementary material The online version of this article (doi:10.1007/s00894-015-2762-6) contains supplementary material, which is available to authorized users. * Raúl Herrera raherre@utalca.cl 1 Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, Casilla 747, Talca, Chile J Mol Model (2015) 21:216 DOI 10.1007/s00894-015-2762-6