A reassessment of Arabidopsis thaliana fructose 1,6 bisphosphate aldolase 6 S-glutathionylation identifies new modified cysteine residues

Redox-dependent post-translational modifications, such as S-glutathionylation, play a key role in regulating plant metabolic enzymes under stress conditions. Here, we studied Arabidopsis thaliana cytosolic 1,6-fructose-bisphosphate aldolase 6 (FBA6) modification by S-glutathionylation. Treatment with the thiol-oxidizing agent diamide inhibited aldolase activity, whereas combined treatment with reduced glutathione and diamide resulted in a stronger inhibition. This enhanced effect was associated with S-glutathionylation as shown by immunoblot analysis using an anti-glutathione antibody. The inhibitory effects were reversible under reducing conditions. Further characterization of FBA6 S-glutathionylation by nanoLC-MS/MS identified five S-glutathionylated Cys residues, including three (Cys130, Cys208, and Cys326) reported here for the first time. Additionally, using protein modeling and nanoLC-MS/MS, we provide evidence for the occurrence of disulfide bridges in the FBA6 structure, including unambiguous confirmation for the existence of a Cys130-Cys173 bond. These findings confirm the redox reactivity of previously uncharacterized FBA6 Cys residues and provide further insights into the redox sensitivity of the enzyme activity. This redox sensitivity may be implicated in the modulation of metabolic and non-canonical functions of this protein under oxidative conditions.