Structural Insights into the Dehydroascorbate Reductase Activity of Human Omega-Class Glutathione Transferases

• Published in: Journal of molecular biology 2012-07, Vol.420 (3), p.190-203
• Main Authors: Zhou, Huina, Brock, Joseph, Liu, Dan, Board, Philip G., Oakley, Aaron J.
• Format: Article
• Language: English
• Subjects: alanine; ascorbic acid; Ascorbic Acid - chemistry; Ascorbic Acid - metabolism; catalytic activity; crystal structure; Crystallography, X-Ray; enzyme mechanism; Enzyme Stability; glutathione; Glutathione - metabolism; glutathione dehydrogenase (ascorbate); Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Humans; induced fit; isozymes; Mutagenesis, Site-Directed; Protein Conformation; reduction; sequence analysis; Serine - chemistry; Serine - genetics; site-directed mutagenesis; structure; transferases
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2012.04.014

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. [Display omitted] ► Omega-class GSTs are vital enzymes in DHA reduction. ► Crystal structures of human omega-class GSTs have been determined. ► The structures provide insight into the mechanism of DHA reduction.