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.

Introduction

Glutathione transferases (GSTs; E.C. 2.5.1.18) are a major class of phase II detoxification enzymes.¹ Their main function is in the conjugation of endogenous or exogenous xenobiotic toxins with electrophilic centers to glutathione (γ-Glu-Cys-Gly; GSH), but several function as GSH peroxidases or as reductases.² The family of cytosolic GSTs comprises different classes including the omega class.³ Omega GST is a class with two different subunits (O1 and O2).4, 5 The omega-class GSTs have been associated directly with several biological processes including the activation of IL-1β⁶ and the modulation of ryanodine receptors.⁷ Although the mechanism has not been elucidated, polymorphisms in the omega-class GSTs have been strongly associated with the age at onset of Alzheimer's and Parkinson's diseases,⁸ and a number of studies have reported associations with a range of disorders including familial amyotrophic lateral sclerosis⁹ and the development of acute childhood lymphoblastic leukemia.¹⁰ Specifically, genetic variation in GSTO2 has been associated with an increased risk of chronic obstructive pulmonary disease,11, 12 urothelial carcinoma¹³ and ovarian cancer.¹⁴

GSTO1-1 and GSTO2-2 show novel thioltransferase activity, as well as dehydroascorbate (DHA) reductase and monomethylarsenate reductase activities.4, 15, 16, 17 We have previously shown that GSTO2-2 has higher DHA reductase (DHAR) activity than GSTO1-1¹⁷ and is considered to be the most active DHAR in mammalian cells. This may be critical in the maintenance of ascorbic acid (AA) levels in the brain since they are dependent on the uptake and subsequent enzymatic reduction of DHA. AA plays a major role in scavenging free radical and specific reactive oxygen species,¹⁸ and the high consumption of oxygen in the brain suggests that a significant capacity to scavenge and detoxify these reactive species is required to prevent oxidative damage. Since the onset of neurological disorders such as Alzheimer's and Parkinson's diseases could be modulated by oxidative stress in the brain, it is important to understand the structure and function of enzymes that play a significant role in regulating redox balance. Given the high level of conservation between human GSTO1-1 and GSTO2-2 sequences (64% identity at the amino acid level), it is likely that they operate by the same mechanism. We have previously determined the crystal structure of GSTO1-1⁴ [Protein Data Bank (PDB) ID: 1EEM]. Previous attempts to express recombinant human GSTO2-2 met with limited success due to poor expression and instability of the protein.⁵ Here, we describe a mutagenesis strategy that was used to improve the solubility and stability properties of GSTO2-2. Non-catalytic cysteine residues predicted to lie on the surface of GSTO2-2 (with potential to destabilize the protein through nonnative disulfide bonds) were mutated to serine, and residues at the C-terminus were deleted in order to stabilize the enzyme. The modified protein retains 70% of the wild-type DHAR activity, and it was sufficiently stable to enable structural and functional characterization. In order to understand the DHAR activity of omega-class GSTs, we determined the structure of a GSTO1-1 mutant in complex with the reaction product AA. Together, the data lead us to propose a catalytic mechanism for DHAR activity in omega-class GSTs and related enzymes.