Phi class glutathione transferases as molecular targets towards multiple-herbicide resistance: Inhibition analysis and pharmacophore design

Abstract

Multiple-herbicide resistance (MHR) is a global threat to weed control in cereal crops. MHR weeds express a specific phi class glutathione transferase (MHR-GSTF) that confers resistance against multiple herbicides and therefore represents a promising target against MHR weeds. Kinetics inhibition analysis of MHR-GSTFs from grass weeds Lolium rigidum (LrGSTF) Alopecurus myosuroides (AmGSTF) and crops Hordeum vulgare (HvGSTF) and Triticum aestivum (TaGSTF) allowed the identification of the acetanilide herbicide butachlor as a potent and selective inhibitor towards MHR-GSTFs. Also, butachlor is a stronger inhibitor for LrGSTF and AmGSTF compared to HvGSTF and TaGSTF from crops. The crystal structure of LrGSTF was determined at 1.90 Å resolution in complex with the inhibitor S-(4-nitrobenzyl)glutathione. A specific 3D pharmacophore targeting the MHR-GSTFs was designed and used to identify structural elements important for potent and selective inhibition. Structural analysis of GSTFs revealed a decisive role of conserved Tyr118 in ligand binding and pharmacophore design. Its positioning is dependent on an outer patch of adjacent residues that span from position 132 to 134 which are similar for both LrGSTF and AmGSTF but different in HvGSTF and TaGSTF. The results presented here provide new knowledge that may be adopted to cope with MHR weeds.

Directed Evolution of Phi Class Glutathione Transferases Involved in Multiple-Herbicide Resistance of Grass Weeds and Crops

“…2.1. Shuffling of Parental GSTF Genes Encoded in A. myosuroides, L. rigidum, T. durum and H. vulgare and Activity Screening Alignment of the parental GSTFs from A. myosuroides, L. rigidum, T. durum and H. vulgare [30,31] showed 88% and 72% sequence homology at the protein and DNA level, respectively. Despite the high homology in primary structures, their kinetics and catalytic properties differ significantly, allowing an interesting research perspective.…”

“…Previous investigations in our lab [30,31] have shown that the parent enzymes display restricted ligandin function and are able to bind a narrow range of pesticides with high affinity. For instance, chloroacetanilide herbicides appear to bind with high affinity among different families of pesticides [31].…”

“…Thereafter, publications on the GST-mediated resistance to multiple herbicides based on an increase in GST activity and/or gene expression were reported [11,[23][24][25][26][27][28]. Notably, detoxifying enzyme levels are greater in domesticated cereal crops than in their competing weeds; thus, these enzymes are largely responsible for differences in herbicide metabolism and selectivity [29][30][31][32][33]. Overexpression of GSTFs has been identified in A. myosuroides and L. rigidum weeds that have demonstrated herbicide tolerance.…”

“…However, the lack of increased detoxifying enzymatic ability in these enzymes due to their kinetic profile has led to the conclusion that they possess a direct regulatory role in cell metabolism by controlling the accumulation of protective flavonoids. The key role of GSTFs in MHR was further highlighted when their inhibition by GST inhibitors helped in restoring herbicide control in A. myosuroides [11,[30][31][32][33]. Furthermore, herbicide resistance of A. myosuroides and Lolium spp.…”

“…In the present work, an in vitro directed evolution approach was implemented via DNA shuffling for homologous recombination of selected gstf genes [30,31] from the cereal crops Triticum durum and Hordeum vulgare, as well as the weeds Alopecurus myosuroides and Lolium rigidum. The work aimed at the creation of a library of detoxifying enzymes with improved catalytic properties and structural stability that could be further utilized in green biotechnology applications.…”