The identification and structural characterization of C7orf24 as γ-glutamyl cyclotransferase: An essential enzyme in the γ-glutamyl cycle

The hypothetical protein C7orf24 has been implicated as a cancer marker with a potential role in cell proliferation. We have identified C7orf24 as γ-glutamyl cyclotransferase (GGCT) that catalyzes the formation of 5-oxoproline (pyroglutamic acid) from γ-glutamyl dipeptides and potentially plays a significant role in glutathione homeostasis. In the present study we have identified the first cDNA clones encoding a γ-glutamyl cyclotransferase. The GGCT gene is located on chromosome 7p14-15 and consists of four exons that span 8 kb. The primary sequence is 188 amino acids in length and is unlike any protein of known function. We crystallized functional recombinant γ-glutamyl cyclotransferase and determined its structure at 1.7 Å resolution. The enzyme is a dimer of 20,994-Da subunits. The topology of GGCT is unrelated to other enzymes associated with cyclotransferase-like activity. The fold was originally classified as "BtrG-like," a small family that only includes structures of hypothetical proteins from Mus musculus, Escherichia coli, Pyrococcus horikoshii, and Arabidopsis thaliana. Since this is the first member of this family with a defined function, we propose to refer to this structure as the γ-glutamyl cyclotransferase fold. We have identified a potential active site pocket that contains a highly conserved glutamic acid (Glu⁹⁸) and propose that it acts as a general acid/base in the reaction mechanism. Mutation of Glu⁹⁸ to Ala or Gln completely inactivates the enzyme without altering the overall fold.