The three-dimensional structure of VvROMT was previously modeled in a closed, catalytically competent dimeric conformation, containing the substrate and the co-substrate S-adenosylmethionine (SAM). The substrate-binding site of VvROMT is composed of hydrophobic residues. Molecular docking revealed that in the catalytically competent binding mode of resveratrol, the molecule is oriented with its 3-OH group towards the sulfonium group of SAM. Specific residues such as F24, F167, F311, and F318 stabilize resveratrol through aromatic interactions, while other hydrophobic interactions involve L117, M121, M171, W258, and M315. Among these, F311 was identified as a critical residue for substrate stabilization and catalytic function, as confirmed by both in silico and experimental analyses. resembles that of other type I OMT substrates that are para methylated, such as isoeugenol and sinapyl alcohol (Clarkia breweri (iso)eugenol O-methyltransferase; PDB codes 3reo, 5cvu), and isoformononetin (Medicago sativa isoflavone-7-O-methyltransferase 8; PDB code 1fp2). However, our predicted binding mode of the substrates 2b and 2f with methylation in meta position resembles that of resveratrol in our VvROMT model, and the meta-methoxy products sinapaldehyde and coniferaldehyde in complex with Lolium perenne caffeic acid O-methyltransferase (PBD codes 3p9i, 3p9k). Table summarizes the activity of VvROMT and the seven variants with active site amino acid substitutions in the conversion of the phenolic acids and phenolic styrenes in comparison to the conversion of two hydroxystilbenes. In the conversion of 1a-d and 2a-d by VvROMT, two main trends were observed: VvROMT converts the phenolic styrenes much faster than the phenolic acids 1. Within the former group of substrates, the activity towards the larger 2c and 2d is higher than towards the smaller 2a and 2b. The discrimination between 1 and 2 can be attributed to differences in their compatibility with the active site. The substrate binding site is highly hydrophobic and does provide little polar surface area for the formation of polar interactions, in particular hydrogen bonds. In the VvROMT structural model, 89% of the surface area of the substrate binding site buried by resveratrol is hydrophobic, as calculated by the tool dr_sasa. Analysis of the electrostatic (Elec) and van der Waals (vdW) energy terms of the binding free energy estimated by the MM-PBSA method in 5 ns molecular dynamics simulations revealed a positive electrostatic energy for the deprotonated acids 1a-d, while negative Elec values were observed for all other ligands (Figure ). The vdW-energy was negative for all ligands and the total free binding energy was close to zero for the deprotonated acid and more negative for all other ligands (Figure ).