# Human glutathione transferases catalyze the reaction between glutathione and nitrooleic acid

## TL;DR

This study shows that human glutathione transferases can speed up the reaction between glutathione and nitrooleic acid, a fatty acid involved in cell signaling and inflammation.

## Contribution

The first demonstration that human GSTs catalyze the reaction between GSH and NO2-FAs.

## Key findings

- hGSTs M1-1 and A4-4 significantly increased the reaction rate between GSH and NO2-OA.
- Crystal structure of hGST M1-1 with the adduct revealed ligand binding in the active site.
- hGST A4-4 formed more interactions with the fatty acid, likely enhancing catalytic activity.

## Abstract

Nitroalkene fatty acids (NO2-FAs) are formed endogenously. They regulate cell signaling pathways and are being developed clinically to treat inflammatory diseases. NO2-FAs are electrophilic and form thioether adducts with glutathione (GSH), which are exported from cells. Glutathione transferases (GSTs), a superfamily of enzymes, contribute to the cellular detoxification of hydrophobic electrophiles by catalyzing their conjugation to GSH. Herein, we evaluated the capacity of five human GSTs (M1-1, M2-2, M4-4, A4-4, and P1-1) to catalyze the reaction between nitrooleic acid (NO2-OA) and GSH. The reaction was monitored by HPLC-ESI-MS/MS, and catalytic activity was detected with hGSTs M1-1 and A4-4. Using stopped-flow spectrophotometry, a 1400- and 7500-fold increase in the apparent second-order rate constant was observed for hGST M1-1 and hGST A4-4, respectively, compared to the uncatalyzed reaction (pH 7.4, 25 °C). The acceleration was in part due to a higher availability of the thiolate. The crystal structure of hGST M1-1 in complex with the adduct was solved at 2.55 Å resolution, revealing that the ligand was bound within the active site, and establishing a foundation to build a model of hGST A4-4 in complex with the adduct. A larger number of interactions between the enzyme and the fatty acid were observed for hGST A4-4 compared to hGST M1-1, probably contributing to the increased catalysis. Altogether, these results show, for the first time, that hGSTs can catalyze the reaction between GSH and NO2-FAs, likely affecting the signaling actions of these metabolites and expanding the repertoire of GST substrates.