engleski

A possible interplay between two conserved Cys residues of auxin-amidohydrolase BrILL2 from Brassica rapa L.

Auxin-amidohydrolases are a group of amidohydrolases from the peptidase M20D family that modulate auxin levels in plants by releasing active plant hormones from their conjugated storage forms. Based on sequence homology, auxin-amidohydrolase BrILL2 from Chinese cabbage (Brassica rapa L.) contains two highly conserved cysteine residues (Cys139 and Cys320). The BrILL2 enzyme heterologously expressed in E. coli and purified via immobilised nickel affinity chromatography preferentially cleaves alaninil-indole-3- propionic acid (IPAala) as a substrate in an enzymatic assay in vitro. We have also determined that the wild type BrILL2 enzyme is active only in the presence of Mn++, as a cofactor, and a reducing agent such as dithiothreitol [1]. In the presence of various reducing agents (DTT, β -mercaptoethanol, reduced glutathione, ascorbic acid and Cys) BrILL2, wt and mutant Cys320Ser retain similar enzymatic activities, whereas they lose activity upon alkylation with J-acetamide or without reducing agents. Site-directed mutagenesis of Cys139 to Ser results in complete inactivation of the enzyme. We confirmed by circular dichroism that, despite mutations, proteins preserve secondary structure. Since enzymes are prone to aggregation in vitro, we have applied gradient SDS-PAGE and Western blot analysis using anti- His antibodies and compared the potential of the wt BrILL2 and the mutants Cys139Ser, Cys320Ser and Cys139, 320Ser for polymerization under reducing conditions vs. oxidizing conditions. Furthermore, possible interaction between above mentioned cysteine residues through intermolecular disulfide bonds formation was analyzed by MALDI-TOF MS. We propose possible enzyme activation in vivo by dissociation of polymers in the presence of natural reducing agents. [1] B. Savić, S. Tomić, V. Magnus, K. Gruden, K. Barle, R. Grenković, J. Ludwig-Muller, B. Salopek-Sondi, Plant Cell Physiol. 2009, 50, 1587-1599.

Auxins ; Auxin-amidohydrolases ; Brassica rapa L.

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