Structural Insights into Omega-Class Glutathione Transferases: A Snapshot of Enzyme Reduction and Identification of a Non-Catalytic Ligandin Site

Abstract

Glutathione transferases (GSTs) are dimeric enzymes containing one active-site per monomer. The omega-class GSTs (hGSTO1-1 and hGSTO2-2 in humans) are homodimeric and carry out a range of reactions including the glutathione-dependant reduction of a range of compounds and the reduction of S-(phenacyl)glutathiones to acetophenones. Both types of reaction result in the formation of a mixed-disulfide of the enzyme with glutathione through the catalytic cysteine (C32). Recycling of the enzyme utilizes a second glutathione molecule and results in oxidized glutathione (GSSG) release. The crystal structure of an active-site mutant (C32A) of the hGSTO1-1 isozyme in complex with GSSG provides a snapshot of the enzyme in the process of regeneration. GSSG occupies both the G (GSH-binding) and H (hydrophobic-binding) sites and causes re-arrangement of some H-site residues. In the same structure we demonstrate the existence of a novel “ligandin” binding site deep within in the dimer interface of this enzyme, containing S-(4-nitrophenacyl)glutathione, an isozyme-specific substrate for hGSTO1-1. The ligandin site, conserved in Omega class GSTs from a range of species, is hydrophobic in nature and may represent the binding location for tocopherol esters that are uncompetitive hGSTO1-1 inhibitors.

Trametes versicolor glutathione transferase Xi 3, a dual Cys‐GST with catalytic specificities of both Xi and Omega classes

“…The crystal structure of an enzyme-substrate complex provided no insight into the reaction mechanism. Indeed, the structure of the mutant HsGSTO1 C32A in complex with glutathionylnitroacetophenone (GS-NAP) revealed the substrate bound in a site too far from the active site to be of catalytic relevance [11].…”

“…Board and coworkers proposed a reaction mechanism for human isoform HsGSTO1 [9]. Indeed, the structure of the mutant HsGSTO1 C32A in complex with glutathionylnitroacetophenone (GS-NAP) revealed the substrate bound in a site too far from the active site to be of catalytic relevance [11]. The enolate is readily protonated to produce the acetophenone.…”

Genomic Analysis of Detoxification Supergene Families in the Mosquito Anopheles sinensis

“…sinensis . The study of Apis cerana provided evidence that the expressions of Omega class GSTs could be induced by various abiotic stresses, which suggested that they play protective roles in counteracting oxidative stresses [ 57 ]. Zeta class GSTs are widely distributed in nature, from plants to animals [ 58 ].…”

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

“…Considering that the active site in GSTO1 contains the highly reactive SH group of Cys32 [2,6], it is likely that it undergoes oxidation and forms mixed disulfide with GSH, at least partly, during preparation of the cytosol. Indeed, the crystal structure of GSTO1 revealed that the active site cysteine residue forms a disulfide with GSH [21].…”

“…This spectrophotometric assay, which is specific for GSTO1, measures the reduction of S-(4-nitrophenacyl)glutathione (4-NPG) into 4-nitroacetophenone in the presence of 2-mercaptoethanol (2-ME). GSTO1 purportedly reacts at its active site cysteine with 4-NPG [4][5][6], thereby releasing the product 4-NAP while forming a GSTO1-glutathione mixed disulfide (Fig. 1).…”