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

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).

Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro

“…Modelling and experiments with plant AATs have provided support for a non‐covalent ternary complex mechanism similar to that found in chloramphenicol acetyltransferase (Morales‐Quintana et al, ; Navarro‐Retamal et al, ; Galaz et al, ). This posits that the active‐site histidine acts as a general base by forming a hydrogen bond between the imidazole NE2 and the alcohol hydroxyl.…”

“…This facilitates nucleophilic attack by the deprotonated hydroxyl at the carbonyl of the acyl-CoA thioester (Kleanthous and Shaw, 1984;Lewendon et al, 1994). It follows from this model 247 Characterization of Atf1p that the active site histidine is expected to be essential for catalysis by the AATs, and indeed this is confirmed by experimental and computational studies of the plant enzymes Navarro-Retamal et al, 2016;Galaz et al, 2013). These models also suggest that the active site aspartic acid is not directly involved in catalysis and does not interact with the histidine in a catalytic dyad, but rather makes other contacts that maintain the structure of the active site .…”

“…Although within this overall fold there are differences in the number and the arrangement of secondary structure elements, the domains generally feature a central β‐sheet region surrounded by α‐helices. Molecular modeling has strongly suggested that plant AATs share this fold (Morales‐Quintana et al, , , ; Navarro‐Retamal et al, ; Galaz et al, ). Substrate access to the buried active site from either face of the enzyme is provided via two linked solvent channels that extend to the protein surface, and in many cases it appears that each of the tunnels is dedicated to one of the individual cosubstrates (Cai et al, ).…”

EngineeringEscherichia colifor the production of butyl octanoate from endogenous octanoyl-CoA

“…2A where domain I, domain II, the crossover loop, the HxxxD motif, and the DFGWG motif are highlighted. Previously, several groups have constructed protein models for AAT enzymes from melon ( Cucumis melo ), mountain papaya fruit ( Vasconcellea pubescens ), and strawberry ( Fragaria x ananassa ) and paired in silico and in vitro analysis to characterise the mechanism of catalysis for these enzymes, as well as the impact of AAT structure on substrate specificity (Morales-Quintana et al, 2011, 2013; Galaz et al, 2013; Navarro-Retamal et al, 2016). From this work, it has been determined that both the alcohol and acyl-CoA substrates access the catalytic His and Asp residues of the HxxxD motif through a solvent channel that runs through the centre of the protein between domains I and II.…”

Energetic differences between non-domain-swapped and domain-swapped chain connectivities in the K2P potassium channel TRAAK

“…MM/ GBSA free energy calculations do not give quantitative estimates for the binding free energies. 16,[25][26][27][28][29][30][31][32][33] It is wise to consider Prime MM/GBSA energy values for comparisons, considering relative free energy values. Therefore, our work could indicate a high prevalence of the DS conformation in TRAAK, which cannot be extended to other K2P channels according to the sequence analysis.…”