Substrate specificity combined with stereopromiscuity in glutathione transferase A4-4-dependent metabolism of 4-hydroxynonenal

Abstract

Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward an endogenously formed toxin.

FIGURE 1

Reaction of racemic HNE with GSH. 1,4-Addition reaction of HNE with GSH as catalyzed by human GSTA4-4, followed by intramolecular cyclization or reduction by NaBH₄.

FIGURE 2

Comparison of stereoselectivity of product formation. The GSHNE diastereomers were prepared by incubating GSH and (A) buffer alone, (B) GSTA4-4, (C) GSTA1-1 GIMFhelix, or (D) GSTA1-1, with racemic HNE and analyzed by LC/MS (ESI+, m/z 464). The corresponding spontaneous reaction (gray line) is also shown for comparison.

FIGURE 3

The overall structure of human GSTA4-4 in complex with 3S,4R-GSDHN (3IK7). The views are aligned perpendicular to the two-fold axis of symmetry for the dimer. (A) Structure emphasizing the active site region within one subunit (cyan: β–strands; gray: α–helices except for the C-terminal and α4-α5 tower regions, which are highlighted in orange throughout all figures). (B) Semi-transparent view of the dimer.

FIGURE 4

Human GSTA4-4 active site. GSTA4-4 is shown in complex with 3S,4R-GSDHN (3IK7, blue ligand). The conserved G-site is toward the lower half of the figure with Y9 shown in cyan. The 4-hydroxyl group is orientated toward R15 while the aldehyde-derived hydroxyl is in proximity of Y212 at the bottom of the H-site. Other important active site residues are labeled and shown as spheres to illustrate the hydrophobic cavity.

FIGURE 5

Position of residues 10 and 220 within human A-class GST active sites. (A) Structural superposition of 3S,4R-GSDHN-bound GSTA4-4 (3IK7, blue ligand) and 3S,4R-GSDHN-bound GSTA1-1 GIMFhelix (3IK9, green ligand) (B) Structural superposition contrasting the side chain of F/P10 and F220. The ball and stick representation of residues from GSTA4-4 and GSTA1-1 GIMFhelix are colored blue and orange, respectively. The ligands are not shown for clarity in (B).

FIGURE 6

C-terminal regions of apo and ligand-bound human GSTA4-4. Structural superposition of apo (1GUM, orange) and 3S,4R-GSDHN-bound GSTA4-4 (3IK7, blue) illustrating the localized structure of the α9-helix regardless of ligand occupancy. Only residues 208-220 are shown for clarity.