Naslov
Possible roles of cysteine residues (Cys139 and Cys320) in the activity of auxin-amidohydrolase BrILL2 from B. rapa

Autori
Smolko, Ana ; Šupljika, Filip ; Martinčić, Jelena ; Jajčanin-Jozić, Nina ; Piantanida, Ivo ; Salopek-Sondi, Branka

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

Izvornik
Chemistry towards Biology 7th Central European Conference - Book of Abstracts / Jarzembek, Krystyna - Katovice, 2014, P17-P17

Skup
Chemistry towards Biology 7th Central European Conference

Mjesto i datum
Katowice, Poljska, 09.09.2014. - 12.09.2014

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Auxin ; amidohydrolase ; auxin homeostasis

Sažetak
1. Introduction Auxin-amidohydrolases (AAHs) from Chinese cabbage (Brassica rapa L.) (BrILL2 and BrIAR3) hydrolase amino acid conjugates (AACs) of plant hormones auxins, thereby releasing free active auxins. The mode of action of free auxins and their accumulation at sites of physiological action are still not well established, but it is evident that AAHs play a role in auxin homeostasis. So far, we established that BrILL2 and BrIA3 preferentially cleave long-chain AACs (IPAala and IBAala), while Arabidopsis AAHs have higher specificity for IAAala. Furthermore, a potential active site of the auxin amidohydrolase BrILL2 was proposed by modelling. AAHs possess two conserved cysteines of which one, Cys 139 is suggested to be a part of the binding pocket that coordinates metal binding. Mn2+ was found to be the only metal ion with cofactor activity, although number and positioning of the cofactor in the binding pocket is still unclear. Herein we investigated the role of Cys residues at positions 139 and 320 in the regulation of the enzyme activity. 2. Results and Discussion The 3D structure of BrILL2 was modeled by using the X-ray structure of A.thaliana IAA-AAH as a template ; the only amidohydrolase crystallized so far with high primary sequence similarity (76%) to BrILL2. The readily recognizable architecture of both AAHs is characterized by two perpendicular domains, with the larger catalytic domain bearing a proposed binuclear metal center, and the smaller ‘‘satellite’’ domain, usually functioning as as a polymerization site. Enzymes, wt and mutants Cys139Ser, Cys320Ser, and Cys139, 320Ser were generated and produced in E. coli as previously described. The preservation of structure of mutants in comparison to wt enzyme was confirmed by circular dichroism. Results of kinetics analysis show that mutant Cys320Ser remains active, but less than wt. Cys139Ser mutation leads to a complete inactivation of the enzyme, probably due to its role in the coordination of one of the Mn2+ ions. Further experiments by isotermal titration calorimetry will give insight into the metal binding and possible role of Cys139.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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