Characterization of the regulation of plant polygalacturonases (PG) by their redox status

master's thesis

• Title (croatian): Karakterizacija regulacije poligalakturonaza (PG) putem njihovog redoks statusa
• Author: Mihaela Šakić
• Mentor: Igor Stuparević (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2024-07-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Polygalacturonases (PGs) are pectin-degrading enzymes that can be found in the primary cell wall where they cleave the α-(1→4) glycosidic bonds of demethylesterified homogalacturonan. PGs have a conserved beta helix fold consisting of three beta sheets with two distinct loops on their surface which surround the active site of the enzymes. Moreover, the PGs structures are characterized by a large number of cysteines that form disulphide bridges. The objective of this internship was to determine the impact of disulphide bridges around PG active site on the activity and processivity of PGLR and ADPG2, by producing mutated versions of these enzymes in a heterologous system Pichia pastoris. The focus was on ADPG2-C156AC332A, and PGLR-D131C-M137C mutants which have been expressed, purified, and characterized regarding their biochemical properties. Our results showed that the optimal substrate for all enzymes is polygalacturonic acid. The highest activity was measured at pH 5, while temperature had little to no effect in the change of activity. The introduction of a new disulphide bond in PGLR-D131C-M137C lead to a decrease in activity but oligoprofiling revealed that there is no difference in processivity compared to the native enzyme. On the other hand, the mutation of two cysteines in ADPG2-C156A-C332A led to complete loss in activity but the enzyme still binds non-methylesterified pectin as shown with MST. Even though these small structural changes led to a change in activity, the global structure of the enzymes was conserved which was confirmed with DSF. Furthermore, a phylogenetic analysis of oxidoreductases in A. thaliana has been done to determine are there possible candidates for in vivo interaction and reduction of disulphide bridges in the cell wall. For this reason, GILT1, GILT2, and TRX2 have been expressed in P. pastoris and in vitro interaction will be tested in the next stages of the project.

• Keywords: pectin; homogalacturonan; polygalacturonase; disulphide bridges; oxidoreductase
• Language: english
• URN:NBN: urn:nbn:hr:159:264176
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Closed access
• Created on: 2024-11-19 13:43:46