Luis Morales-Quintana

Structural characterization and substrate specificity of VpAAT1 protein related to ester biosynthesis in mountain papaya fruit

The aroma in fruits is an important attribute of quality that influences consumers 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.

Structural analysis of the alcohol acyltransferase protein family from Cucumis melo shows that enzyme activity depends on an essential solvent channel

Alcohol acyltransferases (AAT) play a key role in ester biosynthesis. In Cucumis melo var. cantalupensis, AATs are encoded by a gene family of four members (CmAAT1–4). CmAAT1, CmAAT3 and CmAAT4 are capable of synthesizing esters, with CmAAT1 the most active. CmAAT2 is inactive and has an Ala268 residue instead of a threonine which is present in all other active AATs, although the role of this residue is still unclear. The present work aims to understand the molecular mechanism involved in ester biosynthesis in melon fruit and to clarify the importance of the Ala268 residue. First, structural models for each protein were built by comparative modelling methodology. Afterwards, conformational interaction between the protein and several ligands, alcohols and acyl‐CoAs was explored by molecular docking and molecular dynamics simulation. Structural analysis showed that CmAATs share a similar structure. Also, well‐defined solvent channels were described in the CmAATs except for CmAAT2 which does not have a proper channel and instead has a small pocket around Ala268. Residues of the catalytic HxxxD motif interact with substrates within the solvent channel, with Ser363 also important. Strong binding interaction energies were described for the best substrate couple of each CmAAT (hexyl‐, benzyl‐ and cinnamyl‐acetate for CmAAT1, 3 and 4 respectively). CmAAT1 and CmAAT2 protein surfaces share similar electrostatic potentials; nevertheless the entrance channels for the substrates differ in location and electrostatic character, suggesting that Ala268 might be responsible for that. This could partly explain the major differences in activity reported for these two enzymes.

Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.

Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.

In-silico analysis of the structure and binding site features of an α-expansin protein from mountain papaya fruit (VpEXPA2), through molecular modeling, docking, and dynamics simulation studies

AbstractFruit softening is associated to cell wall modifications produced by a set of hydrolytic enzymes and proteins. Expansins are proteins with no catalytic activity, which have been associated with several processes during plant growth and development. A role for expansins has been proposed during softening of fruits, and many fruit-specific expansins have been identified in a variety of species. A 3D model for VpEXPA2, an α-expansin involved in softening of Vasconcellea pubescens fruit, was built for the first time by comparative modeling strategy. The model was validated and refined by molecular dynamics simulation. The VpEXPA2 model shows a cellulose binding domain with a β-sandwich structure, and a catalytic domain with a similar structure to the catalytic core of endoglucanase V (EGV) from Humicola insolens, formed by six β-strands with interconnected loops. VpEXPA2 protein contains essential structural moieties related to the catalytic mechanism of EGV, such as the conserved HFD motif. Nevertheless, changes in the catalytic environment are observed in the protein model, influencing its mode of action. The lack of catalytic activity of this expansin and its preference for cellulose are discussed in light of the structural information obtained from the VpEXPA2 protein model, regarding the distance between critical amino acid residues. Finally, the VpEXPA2 model improves our understanding on the mechanism of action of α-expansins on plant cell walls during softening of V. pubescens fruit. Graphical AbstractHomology model, molecular docking and MD simulations exploring the α-expansin interaction from mountain papaya fruit (VpEXPA2) with two putative ligands. Homology model of VpEXPA2 in surface and cartoon representations, showing the two-domain structure (left). A cellulosic ligand (cellodextrin 8-mer; center) and a hemicellulosic ligand (right) shows different conformation into the open groove of VpEXPA2, and are in agreement with the binding energy differences.

Molecular docking simulation analysis of alcohol acyltransferases from two related fruit species explains their different substrate selectivities

Tropical papaya (Carica papaya) and mountain papaya (Vasconcellea pubescens) fruits are characterised for their strong and particular aroma. The aroma of both fruits is different and dominated by esters, which are synthesised by alcohol acyltransferases (AATs). The ability to produce esters is contrasting, V. pubescens (VpAAT1) being a very active enzyme towards the production of benzyl acetate, whereas C. papaya (CpAAT1) is more active towards the production of ethyl butanoate and methyl butanoate, but not benzyl acetate. In order to understand the mechanism of action at the molecular level, the structural model of CpAAT1 protein was built by comparative modelling. Conformational interaction between the protein and several ligands was carried out by molecular docking. CpAAT1 structure showed two domains connected by a large crossover loop, with a solvent channel in the centre of the structure. CpAAT1 and VpAAT1 proteins showed similar 3D structures, including their catalytic sites, but their solvent channels showed differences in size and shape. CpAAT1 solvent channel is larger, in agreement with its higher selectivity for large acyl-CoA substrates. In addition, the most favourably predicted substrate orientation in CpAAT1 was observed for methanol and butanoyl-CoA, showing a perfect coincidence with the high production rate of methyl butanoate of C. papaya fruit.

Differential expression of ethylene biosynthesis genes in drupelets and receptacle of raspberry (Rubus idaeus).

Red Raspberry (Rubus idaeus) is traditionally classified as non-climacteric, and the role of ethylene in fruit ripening is not clear. The available information indicates that the receptacle, a modified stem that supports the drupelets, is involved in ethylene production of ripe fruits. In this study, we report receptacle-related ethylene biosynthesis during the ripening of fruits of cv. Heritage. In addition, the expression pattern of ethylene biosynthesis transcripts was evaluated during the ripening process. The major transcript levels of 1-aminocyclopropane-1-carboxylic acid synthase (RiACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (RiACO1) were concomitant with ethylene production, increased total soluble solids (TSS) and decreased titratable acidity (TA) and fruit firmness. Moreover, ethylene biosynthesis and transcript levels of RiACS1 and RiACO1 were higher in the receptacle, sustaining the receptacles role as a source of ethylene in regulating the ripening of raspberry.

Understanding the roles of Lys33 and Arg45 in the binding-site stability of LjLTP10, an LTP related to drought stress in Lotus japonicus.

In Lotus japonicus, as in most plants, long-chain fatty acids are important components of cuticular wax, one of the principal functions of which is to act as a barrier to water loss in response to drought stress. It is thought that lipid transfer proteins (LTPs) are involved in the process of cuticle formation. We previously described LjLTP10 as an LTP involved in cuticle formation during acclimation response to drought stress in L. japonicus. The structural model of LjLTP10 had two residues (K33 and R45) in the hydrophobic cavity, although the role of these residues was unclear. In the present work, we investigated the molecular mechanism involved in the transport of lipid precursors in L. japonicus and clarified the importance of the residues K33 and R45. First, in silico site-directed mutagenesis studies were carried out on the LjLTP10 structure. Structural analysis showed that LjLTP10 mutants possess similar structures but their hydrophobic cavities are somewhat different. Unfavorable energies for the interactions of the mutant proteins with different ligands were found by molecular docking and molecular dynamics simulations. We also examined the contributions of energetic parameters to the free energy of the protein–ligand complex using the MM-GBSA method. Results showed that the different complexes present similar, favorable van der Waals interactions, whereas electrostatic interactions were not favored in the mutant structures. Our study indicates that the residues K33 and R45 play a crucial role in maintaining the binding pocket structure required for lipid transport.

Computational study enlightens the structural role of the alcohol acyltransferase DFGWG motif.

AbstractAlcohol acyltransferases (AAT) catalyze the esterification reaction of alcohols and acyl-CoA into esters in fruits and flowers. Despite the high divergence between AAT enzymes, 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 remains 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 differences were revealed in the entire 3D structure. Molecular dynamics simulations and docking studies described unfavorable energies for interaction of the mutant proteins with different substrates, as well as unfavored ligand orientations in the solvent channel. Additionally, we examined the contribution 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 contributions and have unfavorable electrostatic interactions. VpAAT1, Y52F and D381A mutants showed a dramatic reduction 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. Graphical abstractMolecular docking, molecular dynamics (MD) simulations and MM-GBSA free energy calculations were employed to obtain quantitative estimates for the binding free energies of wild type Vasconcellea pubescens alcohol acyltransferase (VpAAT1-WT) and the protein mutants. Left VpAAT1 model structure in cartoon representation showing the solvent channel in the middle of the structure. Center, right Changes in shape and structure in the solvent channel of Y52F and D381A mutant proteins, respectively, compared to WT. The results obtained reveal that the interaction between D381 and Y52 residues is important for the maintenance of solvent channel structure

PrMATE1 Is Differentially Expressed in Radiata Pine Exposed to Inclination and the Deduced Protein Displays High Affinity to Proanthocyanidin Substrates by a Computational Approach

The response to inclination in plants is an attractive and extensively studied biological process. The most commonly held theory proposes a differential growth in stem tissue due to unequal auxin redistribution. Further evidence proposed that flavonoids act as molecular regulators of auxin distribution or flux. It is well known that flavonoids affect auxin distribution, but how intracellular concentration is controlled during the gravitropic response in woody species is still unknown. The MATE family has been widely studied, however the molecular basis of flavonoids transport is still poorly understood. Here, we identified and characterized a full-length cDNA from radiate pine encoding a putative MATE protein. Transcript abundance analysis showed that PrMATE1 is expressed in a spatial and temporal manner in inclined stems. Additionally, PrMATE1 fused to GFP is mainly localized in the vacuolar membrane. A 3D protein model showed 12 transmembrane helices and an open cavity. The protein–ligand interaction was evaluated; favourable binding affinity energies were obtained and suggested epicatechin 3′-O-glucoside as the best putative ligand. In silico mutagenesis analysis was used to identify five residues as important to protein–ligand interaction. The data provide a dynamic view of interaction between PrMATE1 and their putative ligands at the molecular scale.

Comparative in silico study of the differences in the structure and ligand interaction properties of three alpha-expansin proteins from Fragaria chiloensis fruit

Abstract Expansins are cell wall proteins associated with several processes, including changes in the cell wall during ripening of fruit, which matches softening of the fruit. We have previously reported an increase in expression of specific expansins transcripts during softening of Fragaria chiloensis fruit. Here, we characterized three α-expansins. Their full-length sequences were obtained, and through qRT-PCR (real-time PCR) analyses, their transcript accumulation during softening of F. chiloensis fruit was confirmed. Interestingly, differential but overlapping expression patterns were observed. With the aim of elucidating their roles, 3D protein models were built using comparative modeling methodology. The models obtained were similar and displayed cellulose binding module(CBM ) with a β-sandwich structure, and a catalytic domain comparable to the catalytic core of protein of the family 45 glycosyl hydrolase. An open groove located at the central part of each expansin was described; however, the shape and size are different. Their protein–ligand interactions were evaluated, showing favorable binding affinity energies with xyloglucan, homogalacturonan, and cellulose, cellulose being the best ligand. However, small differences were observed between the protein–ligand conformations. Molecular mechanics-generalized Born-surface area (MM-GBSA) analyses indicate the major contribution of van der Waals forces and non-polar interactions. The data provide a dynamic view of interaction between expansins and cellulose as putative cell wall ligands at the molecular scale. Communicated by Ramaswamy H. Sarma