title: Structural insights into the dehydroascorbate reductase activity of human omega-class glutathione transferases.
abstract: The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Huina
authors.initials: H
authors.lastname: Zhou
authors.forename: Joseph
authors.initials: J
authors.lastname: Brock
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Oakley
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry / Q000235:genetics
chemical_list: D012694:Serine; C490476:GSTO1 protein, human; C492569:GSTO2 protein, human; D005982:Glutathione Transferase; D005978:Glutathione; D001205:Ascorbic Acid