Naslov
Amino acid Cys 139 of an auxin amido-hydrolase from Brassica rapa L. plays the key role in the enzyme activity

Autori
Brajlović, Maja ; Kapustić, Ivana ; Savić, Bojana ; Magnus† , Volker ; Salopek Sondi, Branka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
1st International Symposium of Biotech students in Croatia / - Zagreb : Student Association of Biotechnology-Helix, 2009, 20-20

Skup
1st International Symposium of Biotech students

Mjesto i datum
Zagreb, Hrvatska, 24.09.2009. - 26.09.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Brassica rapa L.; auxin amido-hydrolase; BrILL2; Cys139

Sažetak
Auxins are plant hormones involved in many aspects of growth and development. They 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 a metalloenzyme constituted of 444 amino acids and around 48 kDa in size. It hydrolyzes alanine (Ala) conjugates of indole-3-propionic acid (IPA) as a preferable substrate. This enzyme includes 2 Cys residues which are highly conserved in auxin amidohydrolases through different plant species. In order to examine the role of one of them (Cys 139) for enzyme activity, the mutant BrILL2C139S was obtained by using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene) and a correspondig pair of primers. The plasmids pTrcHis2-Topo which comprises a his-tag (wt and mutant) were inserted into E. coli cells, strain BL21(DE3)RIL+, protein overexpression was induced by 0.5 mM IPTG, and proteins were purified by affinity chromatography using Ni-complex linked Sepharose. The enzymatic activities of BrILL2, wt and mutant were tested in the presence of various reduction agents: DTT, β -mercaptoethanol, reduced glutathione, ascorbic acid, and Cys. Progress of the cleavage reaction was monitored by HPLC. Furthermore enzymes were treated with H202, and J-acetamide, and, the changes were monitored by SDS-PAGE. Results showed the loss of activity in mutated enzyme what implies that Cys139 plays the key role for the activity of BrILL2. Bioinformatics analyses of BrILL2 were mostly performed by using on line ExPasy programs (http://www.expasy.ch/). The 3D structure of BrILL2 was modeled by the program PyMOL using the X-ray structure of IAA-aminoacid hydrolase from Arabidopsis thaliana (pdb: 1xmb).

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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