Étude de la S-glutathionylation et d’autres modifications redox d’enzymes du métabolisme primaire chez Arabidopsis thaliana

Reactive oxygen species (ROS) are by-products of metabolism. Some conditions such as biotic and abiotic stresses lead to an increase in ROS levels, which can potentially cause oxidative damages to cell macromolecules including proteins. Indeed, some proteins are sensitive to ROS due to the presence of a thiol group on Cys making these residues prone to oxidation. Cys residues are then targeted by several posttranslational redox modifications including S-glutathionylation. This modification in a disulfide bond formed between a protein Cys residue and the tripeptide glutathione. Protein S-glutathionylation is reversible and protects Cys residues from overoxidation by ROS. This modification is also studied for its signaling roles in redox regulation of plant metabolism. During my PhD, I studied three enzymes from Arabodopsis thaliana primary metabolism: triosephosphate isomerase, alcohol dehydrogenase and enolase. Our hypothesis was that these enzymes could be subject to oxidative modifications, including S-glutathionylation, leading to changes in their enzymatic parameters. These modifications could be part of a metabolic response in plant cells during environmental stress.     Results obtained with triosephosphate isomerase show that its activity is inhibited by S-glutathionylation. Enzyme activity could be recovered by using glutaredoxins that catalyze the reduction of disulfide bonds between the protein and glutathione. The residues Cys127 and Cys218 were found as potential targets of protein S-glutathionylation. We demonstrated by site-directed mutagenesis that mutation of Cys127 cause a large perturbation of the enzyme structure and activity while effects of Cys218 mutation were minimal. Considering literature data, we propose that inhibition of triosephosphate isomerase by S-glutathionylation could take part in redirection of carbon flux through pentose phosphate pathway during stress conditions.     The data on alcohol dehydrogenase suggest that the activity of this enzyme is not affected by S-glutathionylation. However, other modifications such as Cys oxydation and S-nitrosylation can negatively impact the activity of the enzyme. Two Cys (Cys47 and Cys243) were identified as redox sensitive residues. Site-directed mutagenesis suggests that oxidation of Cys47 is the main cause for the decrease in alcohol dehydrogenase activity. Presence of the enzyme cofactor (NADH/NAD+) reduces the inhibitive effect of oxydation and S-nitrosylation, probably by making Cys47 unavailable for the reaction. The possibility of a differential sensitivity to redox modifications due to changes in NADH/NAD+ ratio during hypoxic condition and reoxygenation is discussed.     The research project on enolase has demonstrated the redox-dependent regulatory modification of this enzyme. The enzyme can be S-glutathionylated in vitro in the presence of diamide and deglutathionylated in the presence of glutaredoxin and a glutathione regeneration system. Several Cys residues have been identified as being sensitive to redox changes. A directed mutagenesis approach was used to evaluate the significance of these residues in the activity, structure and redox regulation of enolase. Modification of the enolase by S-glutathionylation could influence carbon fluxes to biosynthetic pathways using phosphoenolpyruvate, particularly in response to stress.

Keyphrases: S-glutathionylation, Redox modifications, Alcohol dehydrogenase