Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from <i>F</i>. <i>x ananassa</i> and Several Alcohol Substrates: A Computational Study

<div><p>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 <i>Fragaria x ananassa</i>, 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 <i>F</i>. <i>x ananassa</i>, 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 <i>Fragaria x ananassa</i> (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 <i>Vasconcellea pubescens</i> (VpAAT1).</p></div

Main distances established between ligands and catalytic residues at the SAAT active site during 30 ns of MD simulations.

<p>(A) Distances between the oxygen atom of the alcohols and the carbonylic carbon of the acetyl-CoA (dHO•••O = C-AcCoA); (B) Distances between the oxygen atom of the alcohols and the Nε of the His157 (dHO•••N_ε-His157). Distances for octanol, hexanol, butanol and benzyl alcohol are represented in black, red, blue and green lines, respectively.</p

Superposition of molecular docking conformations obtained for four alcohol substrates and acetyl-CoA at the back face pocket of SAAT.

<p>Octanol, butanol, hexanol and benzyl alcohol are represented as sticks colored in blue, orange, red and grey, respectively. Acetyl-CoA and His157 are represented in ball and sticks with carbon atoms colored in green and grey, respectively. The hydrogen bonds between N_ε of His157 and hydroxyl group of alcohol substrates are represented as black dashed lines.</p

Comparison of substrate specificity of SAAT and molecular docking energies for the different acetyl-CoA-alcohol-SAAT modeled complexes that could promote the synthesis of the corresponding ester.

<p>Comparison of substrate specificity of SAAT and molecular docking energies for the different acetyl-CoA-alcohol-SAAT modeled complexes that could promote the synthesis of the corresponding ester.</p

Free binding energies and its components calculated from MM-GBSA approach.

<p>Free binding energies and its components calculated from MM-GBSA approach.</p

Main established interactions by Acetyl-CoA at the front face pocket in SAAT

<p>. SAAT is shown in white ribbon representation and acetyl-CoA is represented in balls and sticks with carbon atoms colored in green. Residues His157, Asp166, Asn182 and some positive charged residues (Arg177 and Arg180, which are establishing hydrogen bond interactions with the phosphate groups of the CoA) are shown in licorice representation with carbon atoms colored in grey. Hydrogen bonds between Acetyl-CoA and Arg and Asp residues are depicted as black dashed lines.</p

Image_2_RNAseq, transcriptome analysis and identification of DEGs involved in development and ripening of Fragaria chiloensis fruit.pdf

Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.</p

Data_Sheet_2_RNAseq, transcriptome analysis and identification of DEGs involved in development and ripening of Fragaria chiloensis fruit.xlsx

Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.</p

Data_Sheet_1_RNAseq, transcriptome analysis and identification of DEGs involved in development and ripening of Fragaria chiloensis fruit.xlsx

Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.</p

Data_Sheet_3_RNAseq, transcriptome analysis and identification of DEGs involved in development and ripening of Fragaria chiloensis fruit.xlsx

Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.</p