hrvatski

Uloga cisteina (Cys139 i Cys320) u aktivnosti enzima auksin-amidohidrolaze kineskog kupusa (Brassica rapa L.)

Auksin-amidohidrolaze su grupa enzima koji hidroliziraju aminokiselinske konjugate auksina oslobađajući aktivne spojeve, te na taj način sudjeluju u homeostazi biljnih hormona auksina. U ovom diplomskom radu istražena je uloga dviju visoko sačuvanih aminokiselina –cisteina- u auksin-amidohidrolazi iz kineskog kupusa (Brassica rapa L.), BrILL2, koja hidrolizira konjugat alanina i indol-3-propionske kiseline (IPA-Ala) kao preferirani supstrat. U tu svrhu, proteini, divlji tip i mutanati C139S i C320S, proizvedeni su prekomjernom ekspresijom u E. coli i pročišćeni pomoću Ni-NTA agaroze. Sačuvanost strukture u mutanata s obzirom na divlji tip potvđena je metodom cirkularnog dikroizma. U BrILL2, divljeg tipa i mutanta C320S potvrđena je slična enzimska aktivnost u prisutnosti različitih redukcijskih sredstava. Nedostatak redukcijskog sredstva kao i alkilacija jodoacetamidom uzrokuju gubitak aktivnosti tih enzima. Mutant C320S pokazao se nestabilniji u odnosu na divlji tip BrILL2. Potpuni gubitak enzimske aktivnosti dokazan je u mutanta C139S. Rezultati ukazuje na činjenicu da C139 ima bitnu ulogu u aktivnosti enzima, dok C320 može sudjelovati u regulaciji stabilnosti proteina. Provedena je bioinformatička analiza proteina BrILL2 te predložen model 3D strukture koristeći kao predložak kristalnu strukturu IAA-amidohidrolaze iz Arabidopsis thaliana (pdb: 1xmb). Auxin amidohydrolases are a group of enzymes which hydrolyze the amide bond of conjugated auxins releasing free active auxins, thus participating in homeostasis of plant hormone auxins. Herein we investigate the role of two, highly conserved, cysteine residues in auxin amidohydrolase from Chinese cabbage (Brassica rapa L.), BrILL2, which hydrolyzes alanine conjugate of indole-3-propionic acid (IPA-Ala) as a preferable substrate. The BrILL2 enzymes, wild type and mutants C139S and C320S, were overexpresed in E. coli, and purified by Ni-NTA agarose. Preservation of the structure of the mutants with respect to the wild type was verified by the method of circular dichroism. Similar enzymatic activities of BrILL2, wt and mutant C320S were determined in the presence of various reducing agents. The loss of these enzymes activity was observed upon alkylation with J-acetamide or without reducing agents. Mutant C320S showed lower protein stability in comparison to wild type, while mutated enzyme C139S lost the activity. The results suggest that cysteine 139 is crucial for the activity of BrILL2, while C320 may have a role in the protein stability. Bioinformatics analyses of BrILL2 were performed by using on line ExPasy programs (http://www.expasy.ch/). The 3D structure of BrILL2 was modeled by using the X-ray structure of IAA-aminoacid hydrolase from Arabidopsis thaliana (pdb: 1xmb).

Auksin-amidohidrolaza; cistein (Cys); Brassica rapa L.; BrILL2; enzimska

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