ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione

Abstract

Edited by Ruma BanerjeeGlutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of ␥-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ϳ50% sequence identity. and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either ␥-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed.

The glutathione cycle: Glutathione metabolism beyond the γ‐glutamyl cycle

“…Members of the ChaC family are found from bacteria to human . Humans and mice contain two such enzymes, ChaC1 and ChaC2 . The ChaC1 enzymes have higher catalytic efficiency, are induced to high levels during development and under stress conditions, while the ChaC2 enzymes have lower efficiency and in mammals are constitutive.…”

“…Glutathione biosynthesis in mammals is catalyzed by a GCL that has two subunits, a catalytic subunit, GCLC, and regulatory or modulatory subunit, GCLM. In terms of glutathione degradation, in humans and mouse, there are two ChaC‐like proteins in the cytosol, ChaC1 and ChaC2 . ChaC1 is upregulated during development and during stress conditions while ChaC2 is constitutive and likely to be responsible for the continuous slow degradation of glutathione, where glutathione serves as a reservoir for the rate limiting amino acid, cysteine.…”

Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism

“…ChaC2 showed high structural similarities to yeast glutathione-specific gamma-glutamylcyclotransferase (PDB ID: 5HWI, z-score = 19.2, sequence identity = 38%), human GGCT (PDB ID: 2PN7, z-score = 12, sequence identity = 21%), and human GGACT (PDB ID: 3JUC, z-score = 7.2, sequence identity = 21%). When the structure of the crystallographic ChaC2 dimer with flexible loop2 was superimposed with the structurally similar enzymes, the Glu74 residue in loop2 of ChaC2 was found to be located at the dimer interface and structurally equi-positional to other active-site Glu residues in the structurally similar enzymes [11][12][13] (Figure 3). Thus, we hypothesized that Glu74 plays a critical role not only in the catalytic function but also in the dimerization in a crystal via flexible loop2.…”

“…The GGCT proteins generally have an acid/base Glu in their active site; Glu98 (in human GGCT) [12], Glu82 (in human GGACT) [13], and Glu116 (yeast ChaC2) [11] (Figure 3). To identify the equivalent catalytic Glu residue of ChaC2, we carefully investigated the active site residues of ChaC2 with other GGCT enzymes ( Figure 5B).…”

Transcriptomic Profiling of MDA-MB-231 Cells Exposed to Boswellia Serrata and 3-O-Acetyl-Β-Boswellic Acid; ER/UPR Mediated Programmed Cell Death

“…While discussing all the changes in the transcriptome initiated by 3-OAβBA and BSE are beyond the scope of this paper, noteworthy is the rise in CHAC1, which is involved in the degradation of glutathione (62,63) reported to occur in parallel to rise of ATF4-ATF3-CHOP PERK and the phosphorylation of EIF2α, where its rise creates vulnerability of cancer cells to the losses of glutathione associated with radiation and oxidative insult (64, 65) also rendering losses on glutathione detoxification systems (66).…”