Biokemijska i funkcionalna karakterizacija enzima auksin-amidohidrolaze BrILL2 iz kineskoga kupusa (Brassica rapa L. ssp. pekinensis)

doctoral thesis

• Title (english): Biochemical and functional characterization of an auxin amidohydrolase enzyme BrILL2 from Chinese cabbage (Brassica rapa L. ssp. pekinensis)
• Author: Ana Smolko
• Mentor: Branka Salopek-Sondi (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Branka Salopek-Sondi (član povjerenstva)
• Committee member: Nataša Bauer (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2019-09-17, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Auksin-amidohidrolaza ILL2 biljni je enzim iz porodice metalopeptidaza M20, koji specifično hidrolizira amidnu vezu u inaktivnim konjugatima auksina s aminokiselinama oslobađajući slobodan aktivan auksin. Homolog ILL2 iz kineskoga kupusa (Brassica rapa L. ssp. pekinensis) biokemijski je okarakteriziran te je objašnjena uloga očuvanih aminokiselinskih ostataka Cys139, Cys320, Phe385, Arg295 i Met367 u enzimskoj aktivnosti i stabilnosti proteina te vezanju metala. Predložena je aktivacija BrILL2 in vivo disocijacijom oligomernih formi enzima uz prisustvo prirodnih reducensa. Supstratna specifičnost enzima BrILL2 prema konjugatima dugolančanih auksina, kao i hidroliza tih konjugata in vivo potvrđena je koristeći tzv. „feeding“ eksperimente. Auksinski metabolom po prvi put je opisan za mutante s disfunkcionalnim amidohidrolazama. Utvrđena je preraspodjela slobodnog auksina u biljkama po tretmanu konjugatima dugolančanih auksina, izražena kroz pojačanu konjugaciju auksina te oksidaciju suviška auksina. Akumulacija auksina u korijenu klijanaca reporterske linije uročnjaka (Arabidopsis thaliana) DR5rev::GFP ukazuje na rani odgovor biljaka na tretman auksinima.

Abstract (english)

Auxin-amidohydrolase ILL2 is a plant enzyme from the M20 metalopeptidases family, which specifically hydrolyses amide bond in inactive auxin conjugates with amino acids thereby releasing free active auxin. Homologue ILL2 from Chinese cabbage (Brassica rapa L. ssp. pekinensis) was biochemically characterized and the role of preserved amino acid residues Cys139, Cys320, Phe385, Arg295 and Met367 in enzymatic activity, protein stability and metal binding was explained. Activation of BrILL2 was proposed by dissociation of oligomeric forms of the enzyme in vivo in presence of natural reductants. Substrate specificity of BrILL2 enzyme towards long-chain auxin conjugates, as well as hydrolysis of these conjugates in vivo, has been confirmed using feeding experiments. Auxin metabolome was described for mutants with dysfunctional amidohydrolases. A change in auxin distribution was determined in plants upon treatment with long-chain auxin conjugates, expressed through enhanced auxin conjugation, and oxidation of surplus of auxin. Accumulation of auxin in the root of Arabidopsis thaliana reporter line DR5rev::GFP suggests an early plant response to auxins’ treatment.

• Keywords: auksin-amidohidrolaza; Brassica rapa; auksinska homeostaza
• Keywords (english): auxin amidohydrolase; Brassica rapa; auxin homeostasis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:487660
• Study programme: Title: Doctoral study in chemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija (doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija)
• Type of resource: Text
• Extent: 143 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2021-09-20
• Created on: 2019-09-27 09:30:07