Protein-Ligand Fishing for Biologically Active Natural Products Using Glutathione Transferases.

Screening for natural products which bind to proteins has been used to identify ligands of the plant-specific glutathione transferase (GST) tau (U) and phi (F) classes, that are present in large gene families in crops and weeds, but have largely undefined functions. When expressed as recombinant proteins in these proteins have been found to tightly bind a diverse range of natural product ligands, with fatty acid-and porphyrinogen-derivatives associated with GSTUs and a range of heterocyclic compounds with GSTFs. With an interest in detecting the natural binding partners of these proteins , we have expressed the two best characterized GSTs from (), GSTF2 and GSTU19, as -tagged fusion proteins Following transient and stable expression in Nicotiana and Arabidopsis, respectively, the GSTs were recovered using Strep-Tactin affinity chromatography and the bound ligands desorbed and characterized by LC-MS. GSTF2 predominantly bound phenolic derivatives including -glutathionylated lignanamides and methylated variants of the flavonols kaempferol and quercetin. GSTU19 captured glutathionylated conjugates of oxylipins, indoles, and lignanamides. Whereas the flavonols and oxylipins appeared to be authentic ligands, the glutathione conjugates of the lignanamides and indoles were artifacts formed during extraction. When tested for their binding characteristics, the previously undescribed indole conjugates were found to be particularly potent inhibitors of GSTU19. Such ligand fishing has the potential to both give new insight into protein function as well as identifying novel classes of natural product inhibitors of enzymes of biotechnological interest such as GSTs.