Naslov
BrILL2, auksin amidohidrolaza iz kineskog kupusa (Brassica rapa L.)
(BrILL2, auxin amidohydrolase from Chinese cabbage (Brassica rapa L.))

Autori
Savić, Bojana ; Tomić, Sanja ; Magnus, Volker ; Salopek-Sondi, Branka

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

Izvornik
Zbornik sažetaka / Zahradka, Ksenija ; Plohl, Miroslav ; Ambriović-Ristov, Andreja - Zagreb : Institut Ruđer Bošković, 2008, 74-74

ISBN
978-953-6690-78-7

Skup
50 godina molekularne biologije u Hrvatskoj

Mjesto i datum
Zagreb, Hrvatska, 20.11.2008. - 21.11.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
BrILL2; auksin amidohidrolaza; kineski kupus; Brassica rapa L.
(BrILL2; auxin amidohydrolase; Chinese cabbage; Brassica rapa L.)

Sažetak
Auksini, kao što su indol-3-octena kiselina (IAA), indol-3-propionska kiselina (IPA) i indol-3-maslačna kiselina (IBA), su biljni hormoni uključeni u mnoge procese rasta i razvoja biljke. Njihove endogene koncentracije precizno su regulirane reverznom konjugacijom s aminokiselinama. Iz laboratorija prof. Jutte Ludwig- Müller (1) dobiven je uklonirani gen za jednu od auksin-amidohirolaza iz kineskog kupusa (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt) uključenih u ovaj regulatorni mehanizam. Rekombinantni protein dobiven je heterolognom ekspresijom u E.coli. Nakon čišćenja afinitetnom kromatografijom na Ni-NTA koloni, hidrolitička aktivnost proteina testirana je na IAA, IPA i IBA konjugatima alanina (Ala). Aktivnost je opažena u prisutstvu kofaktora Mn++ i reducirajućih sredstava kao što je dithiotreithol. Tijek enzimatske reakcije praćen je HPLC-om. Najveća brzina hidrolize opažena je kod IPA-ala, nakon čega slijede IBA-ala i IAA-ala. Model 3D strukture BrILL2 i potencijalno vezno mjesto za Mn++ predviđeno je korištenjem programa Modeller9v2, po predlošku strukture dobivene X-zrakama IAA amidohidrolaze iz Arabidopsis thaliana (PDB_id 1XMB). Programom AutoDock 3.05 predviđena su vezna mjesta za ligande IBA-ala, IPA-ala i IAA-ala. Iz dobivenih rezulatata, pomoću programa AMBER9 predložen je mehanizam hidrolize IPA-ala. (1). Schuller, A., and Ludwig-Müller, J. (2006), New Phytol. 171, 145-158. 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.

Izvorni jezik
Hrvatski

Znanstvena područja
Fizika, Biologija

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