engleski

The potential role of Cys (Cys 139) in the activity of BrILL2, an auxin amido-hydrolase from Brassica rapa L.

Auxins are plant hormones involved in many aspects of growth and development, from cell division and elongation to the formation of buds, roots, and fruit. Auxins exist as free auxins (active form) and conjugated with amino acids (storage forms). One of the ways plants control internal levels of auxins is through reversible formation of auxin conjugates. Auxin amidohydrolases are a group of enzymes which hydrolyze the amide bond of conjugated auxins releasing free active auxins. Herein we investigate the auxin-amidohydrolase from Chinese cabbage (Brassica rapa L.), BrILL2. It is named thus because it shows homology with ILL2, an auxin-amidohydrolase previously identified in Arabidopsis thaliana. BrILL2 is a metalloenzyme constituted of 444 amino acids and around 48 kDa in size. According to prediction programs that analyze the amino acid sequence of proteins, this enzyme is probably located in the endoplasmatic reticulum. It hydrolyzes alanine (Ala) conjugates of indole-3-propionic acid (IPA) as a preferable substrate. This enzyme includes 2 Cys which are highly conserved in auxin amidohydrolases through different plant species. One of them (Cys 139) might be substantial for enzyme activity. To obtain enough protein for experiments we used the previously cloned gene for BrILL2 in the expression vector pTrcHis2-Topo which comprises a his-tag. Furthermore, the mutant BrILL2 C139S was obtained by using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene) and a correspondig pair of primers. The plasmids (wt and mutant) were inserted into E. coli cells, strain BL21(DE3)RIL+ by electroporation. Protein overexpression was induced by 0.5 mM IPTG. Proteins were purified by affinity chromatography using Ni-complex linked Sepharose. The enzymatic activities of purified BrILL2, and the mutant BrILL2 C139S were tested. We examined the presence of various reduction agents: DTT, β -mercaptoethanol, reduced glutathione, ascorbic acid, and Cys on enzyme activity. Furthermore enzymes were treated with H202, and J-acetamide, and, the changes were monitored by SDS-PAGE and by comparing hydrolytic activity with that of the non-mutagenized protein. Progress of the cleavage reaction was monitored by HPLC.

Brassica rapa L.; Auxin amidohydrolase; Cys139 residue

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