engleski

Auxin amidohydrolases from Brassica rapa cleave conjugates of indole propionic and indole butyric acid as preferable substrates: A biochemical and modeling approach

Two auxin-amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage (Brassica rapa L. ssp. pekinensis (Lour.) Hanelt) were produced by heterologous expression in E. coli, purified, and screened for activity towards different amino acid (alanine, beta-alanine, aspartate) conjugates with the auxins indole-3-acetic acid (IAA, indole-3-propionic acid (IPA) and indole-3-butyric acid (IBA). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics was measured for BrILL2 and the obtained parameters suggested similar binding affinities for long-chained auxin-amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. The two conjugates IPA-Ala and IBA-Ala showed higher root growth inhibition of Brassica seedlings in comparison to IAA-Ala, indicating cleavage of these conjugates also in vivo. A model of BrILL2 was generated using the X-ray structure of Arabidopsis thaliana IAA-amino acid conjugate hydrolase as a template. The metal binding and substrate binding sites are proposed. This is the first report on auxin amino acid conjugate hydrolases from a dycotyledonous plant species, which cleaves longer chain auxins with preference over IAA conjugates. Because of the high activities found for these enzymes, some other possible functions will be discussed.

Auxin amidohydrolase; Auxin amino acid conjugate; Brassica rapa; Chinese cabbage; Enzyme kinetics; Modeling

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