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Insight into structure and function of the plant enzyme, auxin amido-hydrolase

Auxins, such as indole-3-acetic acid (IAA), indole-3-propionic acid (IPA) and indole-3-butyric acid (IBA), are plant hormones involved in many aspects of growth and development. Their endogenous levels are tightly regulated by reversible conjugation with amino acids. One of the auxin-amidohydrolases included in this regulatory mechanism, BrILL2, was cloned from Chinese cabbage (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt), and the gene was kindly provided to us by prof. Jutta Ludwig-Müller (1). The recombinant, his-tagged, protein was produced by heterologous expression in E. coli. Following affinity purification on a Ni-NTA column, the protein was tested for hydrolytic activity towards the alanine (Ala) conjugates of IAA, IPA and IBA. This was only observed in the presence of Mn++, as a cofactor, and a reducing agent such as dithiothreitol. Progress of the cleavage reaction was monitored by HPLC. IPA-ala was hydrolyzed at the highest rate, followed by IBA-ala and IAA-ala, in this order. The 3D structure of Br-ILL2 was modeled by the program Modeller9v2, in analogy to the X-ray structure of an IAA-amino acid hydrolase from Arabidopsis thaliana (PDB_id 1XMB), and the Mn++ binding site was determined. The ligands, IBA-Ala, IPA-ala and IAA-Ala were docked into the protein using the AutoDock 3.05 program. The most populated binding site was selected as the most probable one. Subsequently the solvated complex was simulated using the program AMBER9, and according to the results a possible scenario for IPA-ala hydrolysis is proposed. (1). Schuller, A., and Ludwig-Müller, J. (2006), New Phytol. 171, 145-158.

Auxin-amidohydrolase; BrILL2; Auxin-amino acid conjugates; Brassica rapa L.

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