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Thermodynamics of ligand binding to auxin amidohydrolase

Recent discoveries in plant hormone signaling have greatly increased the interest for this exciting and highly complex field of research. Auxins, with the most abundant representative indole–acetic acid (IAA), are a group of plant hormones that in very small concentrations regulate ubiquitin- mediated degradation of transcription regulators. They are stored and transported in the form of conjugates, mostly with amino acids, and their release is controlled by a group of enzymes known as auxin amidohydrolases, members of the M20 family of metallopeptidases. Structurally, they are characterized by having two perpendicular domains, and its members have been implicated in numerous biochemical processes and found in all organisms sequenced to date. Kinetic studies on auxin amidohydrolase from Brassica rapa (BrILL2) showed that it performs the hydrolysis of alanine conjugated indole-acetic acid (IAA–Ala) and its higher analogues, indole-propionic acid (IPA–Ala) and indole-butyric acid (IBA–Ala). Also the kinetic parameters (namely the Michaelis constant) suggest stronger binding affinity with the higher analogues (IPA–Ala and IBA–Ala), while the catalytic constant is greatest with IPA–Ala. We carried out molecular dynamics simulations using a semi-empirical force-field to study structural and thermodynamic changes upon ligand binding to BrILL2. We observed a conformational change in a 10 ns simulation, described by a large-scale movement of the satellite (non-catalytic) domain, and traced key interactions that lead to the change, in support of published mutation and kinetic analyses. Also, we calculated binding affinities for four substrates in two possible binding modes, in order to explain the observed selectivity to higher auxin conjugates and propose a possible route for the reaction.

molecular dynamics ; thermodynamics ; hydrolase ; auxin

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