Abstract
Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT) (EC 2.3.1.84) catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD) that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT) using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD) simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This mechanism is in agreement with previous results, obtained in our group, in alcohol acyltransferase from Vasconcellea pubescens (VpAAT1).
Highlights
During fruit ripening, a number of morphological and physiological changes occur converting a green immature fruit into a more attractive to consumers with several organoleptic properties and full ripen fruit
The initial pair-wise alignment of the protein template (PDB ID code: 2BGH) against the target sequence (SAAT) with 32.4% of sequence identity and 50.6% of sequence similarity was optimized manually, incorporating information about secondary structure of the BAHD superfamily, specially in relation to the conserved motifs HXXXD and DFGWG, which were spatially restricted during modeling to avoid any distortion from their initial conformations (See S1 Fig)
The in silico model obtained for SAAT allowed us to dissect the most important structural characteristics of this alcohol acyltransferase (AAT) member of the BAHD family
Summary
A number of morphological and physiological changes occur converting a green immature fruit into a more attractive to consumers with several organoleptic properties and full ripen fruit. The specific aroma of each fruit is generated by the accumulation of a pool of volatile compounds, where the most abundant in Fragaria genus are esters compounds [2,3]. Esters are generated by an esterification reaction, between an acyl-CoA and one alcohol molecule, which is catalyzed by the enzyme alcohol acyltransferase (AAT; EC 2.3.1.84). Plant AATs are monomeric enzymes with a molecular mass between 48 to 55 kDa [1,5]. These enzymes are cytosolic because they do not present destination sequences to organelles or to excretion’s route, it has been proved that their activities are strictly related to the presence of a Histidine residue in the conserved HXXXD motif [11]
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