AbstractBrILL2 is catalytically the most efficient auxin amidohydrolase from Brassica rapa, playing a key role in auxin metabolism by catalyzing its release from amino acid conjugates. Auxins, with the most abundant representative indole‐acetic acid ([1H‐indol‐3‐yl]‐acetic acid, IAA), are a group of plant hormones that in very small concentrations regulate ubiquitin‐mediated degradation of transcription regulators. Kinetic studies on BrILL2 showed that it hydrolyzes alanine conjugates of IAA and of its larger analogues, indole‐propionic acid (3‐[1H‐indol‐3‐yl]‐propionic acid, IPA) and indole‐butyric acid (4‐[1H‐indol‐3‐yl]‐butyric acid, IBA). Structurally, BrILL2 belongs to the largest known family of metallopeptidases (M20) that share a recognizable 3D structure, characterized by two perpendicular domains. Its members have been implicated in numerous biochemical processes and have been found across all species sequenced to date. Here, molecular dynamics simulations were carried out to study structural and thermodynamic properties of ligand binding to BrILL2. A conformational change was captured in multiple copies of 10 ns long simulations, described by a rigid body movement of the two domains, and its associated key interactions between residues were examined. For the three substrates, complexes in two possible binding modes were recreated, along with a single binding mode for the putative substrate tryptophanyl–alanine (Trp–Ala), which were subsequently simulated in multiple copies of 10 ns long simulations. Thermodynamic calculations were used to assess their binding affinities and explain the selectivity toward the longer ligands. Based on the results, a possible route for the reaction is proposed. Copyright © 2011 John Wiley & Sons, Ltd.