Thesis
English
ID: <
10670/1.d8pwav>
Abstract
Plant invertases (Invs) hydrolyze irreversibly sucrose into fructose and glucose. Based on their pH optima and subcellular localization, Invs are categorized into three groups: alkaline and neutral invertase (A/N-Inv), vacuolar invertase (VI), and cell wall invertase (CWI). The goal of our study was to better understand mechanisms involved in the molecular regulation of a VI from Solanum lycopersicum (VINV) at post-translational levels. The VINV cDNA was cloned and heterologously expressed in Pichia pastoris. After purification, the biochemical characterization was performed and showed comparable results with those obtained previously for other characterized Invs. The three-dimensional structure of VINV was solved by X-ray crystallography to 2.75 Å resolution and it was the first structure of a plant VI described so far. Mutations experiments allowed to identify important amino acids: the nucleophile, the acid/base catalyst, the transition-state stabilizer and a residue that modulate pKa of the acid/base catalyst. Moreover, the regulation of VINV at different post-translational levels was studied. N-glycosylation of recombinant VINV seems to be important for structure stability. VINV activity can also be modulated by specific proteinaceous inhibitor. A functional genomics approach was used, and a putative vacuolar invertase inhibitor (SolyVIF) of S. lycopersicum was identified in the Solanaceae data bank. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli Rosetta gami (DE3). Recombinant protein was purified and characterized.