Defining the cytosolic pathway of glutathione degradation in Arabidopsis thaliana: role of the ChaC/GCG family of gamma-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase

作者：Kumar Shailesh; Kaur Amandeep; Chattopadhyay Banani; Bachhawat Anand K^*

来源：Biochemical Journal, 2015, 468(1): 73-85.

DOI：10.1042/BJ20141154

• gamma-glutamyl cycle
• gamma-glutamyl cyclotransferase
• Cys-Gly peptidase
• ChaC
• glutathione LAP1

Glutathione homoeostasis is critical to plant life and its adaptation to stress. The gamma-glutamyl cycle of glutathione biosynthesis and degradation plays a pre-eminent role in glutathione homoeostasis. The genes encoding two enzymatic steps of glutathione degradation, the gamma-glutamyl cyclotransferase (GGCT; acting on gamma-glutamyl amino acids) and the Cys-Gly dipeptidase, have, however, lacked identification. We have investigated the family of GGCTs in Arabidopsis thaliana. We show through in vivo functional assays in yeast that all threemembers of theChaC/GCG subfamily show significant activity towards glutathione but no detectable activity towards gamma-glutamyl methionine. Biochemical characterization of the purified recombinant enzymes GGCT2; 2 and GGCT2; 3 further confirmed that they act specifically to degrade glutathione to yield 5-oxoproline and Cys-Gly peptide and show no significant activity towards gamma-glutamyl cysteine. The K-m for glutathione was 1.7 and 4.96 mM for GGCT2; 2 and GGCT2; 3 respectively and was physiologically relevant. Evaluation of representative members of other subfamilies indicates the absence of GGCTs from plants showing significant activity towards gamma-glutamyl-amino acids as envisaged in the classical gamma-glutamyl cycle. To identify the Cys-Gly peptidase, we evaluated leucine aminopeptidases (LAPs) as candidate enzymes. The cytosolic AtLAP1 (A. thaliana leucine aminopeptidase 1) and the putative chloroplastic AtLAP3 displayed activity towards Cys-Gly peptide through in vivo functional assays in yeast. Biochemical characterization of the in vitro purified hexameric AtLAP1 enzyme revealed a K-m for Cys-Gly of 1.3 mM that was physiologically relevant and indicated that AtLAP1 represents a cytosolic Cys-Gly peptidase activity of A. thaliana. The studies provide new insights into the functioning of the gamma glutamyl cycle in plants.

• 出版日期2015-5-15