Structures of yeast glutathione-S-transferase Gtt2 reveal a new catalytic type of GST family

Abstract

Glutathione-S-transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand-bound forms, at 2.23 A, 2.20 A and 2.10 A, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues--tyrosine, serine and cysteine--distinguishes it from all other cytosolic GSTs of known structure. Site-directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino-terminus of helix-alpha1 because of stereo-hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.

Figure 1

Overall structure of Gtt2. (A) Schematic representation of the Gtt2 homodimer. GTS molecules are shown as stick models and coloured according to their atom type (C, green; S, yellow; O, red). The G-site and H-site of subunit B are indicated by arrows. (B) The GTS-bound Gtt2 monomer, with the two domains in cyan and red. (C) Hydrogen bonds between GTS and Gtt2. (D) Superposition of apo-bound (light blue) and GTS-bound Gtt2 (light orange) showing the different position of the loop after helix-α2 upon GTS binding. Cys, cysteine; G-site, GSH-binding site; GTS, glutathione sulphonate; H-site, hydrophobic substrate binding site; His, histine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

Figure 2

Residues crucial for Gtt2 activity. (A) The sulphur atom of GTS is located over the amino-terminus of helix-α1, at Gly 27. Ser 129 and His 133 form a ‘clamp' to grip water molecule Wat 235, which forms a hydrogen bond with the sulphur atom. The electron density maps of GTS, Wat 235, Ser 129 and His 133 (2F_o−F_c map) were contoured at 1.5 σ. (B) The active site of GSH in the GSH-bound form is located close to that of GTS in the GTS-bound form. (C) The active site of apo-form Gtt2. A water molecule (Wat 243′) locates at about the same position as Wat 235 in the GTS-bound structure, whereas several water molecules take the place of GTS. (D) The relative activity of the wild-type and seven Gtt2 mutants. GSH, glutathione; GTS, glutathione sulphonate.

Figure 3

Conservation of the amino-terminal regions of various types of GST. Sequences of tyrosine, serine and cysteine types are from Protein Data Bank (www.rcsb.org; PDB codes 1TU7, 2A2R, 1XW6, 1GSU, 2CVD, 2GSQ, 1VF1, 1GUL, 2VO4, 1OYJ, 1AXD, 1GNW, 1PN9, 1R5A, 2C3N, 2LJR, 1E6B, 2CZ2, 2GDR, 1N2A, 1PMT and 1EEM). Sequences of the atypical type are downloaded from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov). Codes in parentheses are NCBI accession numbers: Saccharomyces cerevisiae Gtt2, Schizosaccharomyces pombe GST I (Q9Y7Q2), S. pombe GST II (O59827), Burkholderia cenocepacia PC184 GST (YP_002095143), Hahella chejuensis KCTC 2396 GST (YP_437484), Methylibium petroleiphilum PM1 GST (YP_001020104), Alpha proteobacterium HTCC2255 GST (ZP_01450455) and Bradyrhizobium sp. ORS278 GST (YP_001202246). GTS, glutathione sulphonate.