Decoding how enzymes process healing fats

Nitroalkene fatty acids are fascinating molecules: they naturally form in our bodies and act as powerful regulators of signalling with anti-inflammatory and protective properties on cells. Understanding how they are metabolised is crucial to both fundamental biology and drug development, since some of these molecules are already being tested as treatments for inflammatory and cardiovascular diseases. Until recently, it was thought that their reaction with glutathione, a small molecule essential for cellular detoxification, happened spontaneously, without help from enzymes.

In an article recently published in the Journal of Biological Chemistry, an international team of researchers has now shown that members of the human glutathione transferase (GST) enzyme family can catalyse this reaction with remarkable efficiency. Using ambient temperature macromolecular crystallography at Diamond Light Source’s VMXi beamline, the team solved the crystal structure of one of these enzymes bound to the reaction product, providing unprecedented insight into the molecular details of this transformation.
 

Figure: Crystal structure of hGST M1-1 in complex with the GS-10-NO2-OA adduct.A, the hGST M1-1 dimer with the GS-10-NO2-OA adduct in the active site obtained by X-ray crystallography (PDB 8VOU) is shown from two different angles (90° rotation along the y-axis). The protein backbone is represented as a cartoon in gray and the ligand is represented as sticks (carbon cyan, oxygen red, nitrogen blue, and sulphur yellow).

Nitroalkene fatty acids (NO₂-FAs) are electrophilic lipids produced during digestion and inflammation. Their ability to form reversible bonds with proteins underpins many of their biological effects, influencing pathways involved in inflammation, stress response, metabolism, DNA repair and more. A central detoxification route involves their reaction with glutathione (GSH), forming new products of the chemical reaction, that are exported from cells and eventually excreted.

The team worked with nitrooleic acid (NO₂-OA), a model NO₂-FA currently in clinical development. They tested five human cytosolic GST isoforms for their ability to catalyse its reaction with GSH. Previous studies had not detected any catalytic effect, but the researchers hypothesised that this might be due to experimental limitations rather than biological absence.

They found that hGST M1-1 and hGST A4-4 markedly accelerated the formation of the NO₂-OA–GSH adduct. Stopped-flow kinetics revealed rate enhancements of 1,400-fold and 7,500-fold respectively compared to the uncatalysed reaction. This acceleration stems partly from the enzymes lowering the pKa of GSH, increasing the proportion of its reactive thiolate form, but also from specific interactions that orient and stabilise the reaction intermediates.

To reveal these interactions, the team crystallised hGST M1-1 with the reaction product and collected diffraction data on VMXi, Diamond’s state-of-the-art in situ macromolecular crystallography beamline. VMXi enables rapid screening and high-quality structure determination directly from crystallisation plates. The structure, solved at 2.55 Å resolution, clearly showed the adduct in the enzyme’s active site, forming key hydrogen bonds and hydrophobic contacts that explain how the enzyme catalyses the reaction. This structural model also guided computational modelling of the A4-4 isoform, revealing differences in the fatty acid-binding pocket that likely account for its higher activity.

This work expands the known repertoire of GST substrates and provides a new perspective on how these abundant enzymes might modulate bioactive lipid levels inside cells. The findings have implications for understanding drug metabolism and the design of therapeutic strategies using NO₂-FAs. For example, the ability of GSTs to accelerate adduct formation could influence the duration and potency of NO₂-FA-based treatments.

To find out more about the VMXi beamline, please contact the Principal Beamline Scientist Michael Hough: michael.hough@diamond.ac.uk.