Structural Insights into the Dehydroascorbate Reductase Activity of Human Omega-Class Glutathione Transferases
| Autor: | Aaron J. Oakley, Philip G. Board, Joseph Brock, Dan Liu, Huina Zhou |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Alanine
biology Protein Conformation Chemistry Stereochemistry Active site Ascorbic Acid Glutathione Reductase Crystallography X-Ray Ascorbic acid Isozyme chemistry.chemical_compound Biochemistry Structural Biology Enzyme Stability Mutagenesis Site-Directed Serine biology.protein Humans Transferase Binding site Molecular Biology Glutathione Transferase |
| Zdroj: | Journal of Molecular Biology. 420:190-203 |
| ISSN: | 0022-2836 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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