N2‐Acetylornithine deacetylase functions as a Cys‐Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana

SUMMARY Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ‐glutamyl cyclotransferase, and γ‐glutamyl peptidase, which release cysteinylglycine (Cys‐Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys‐Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys‐Gly. The Arabidopsis cgp1 mutant had lower Cys‐Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys‐Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O‐acetylserine (OAS) and the decrease in sulfur‐containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2‐acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys‐Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys‐Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism. Significance Statement In this study, we identified At4g17830 (CGP1) as a Cys‐Gly dipeptidase that functions in the glutathione (GSH) degradation pathway and revealed its importance in plant metabolism and growth, especially under sulfur starvation. Notably, CGP1 has been reported as an N2‐acetylornithine deacetylase (NAOD) that participates in the ornithine biosynthesis pathway; therefore, our findings imply that CGP1 is a bifunctional enzyme that plays vital roles in both sulfur and nitrogen metabolism.