Investigation of the target-site resistance of EPSP synthase mutants P106T and T102I/P106S against glyphosate

Abstract

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) catalyzes the reaction involved in the production of amino acids essential for plant growth and survival. Thus, EPSPS is the main target of various herbicides, including glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. However, punctual mutations in the EPSPS gene have led to glyphosate resistance in some plants. Here, we investigated the mechanism of EPSPS resistance to glyphosate in mutants of two weed species, Conyza sumatrensis (mutant, P106T) and Eleusine indica (mutant, T102I/P106S), both of which have an economic impact on industrial crops. Molecular dynamics (MD) simulations and binding free energy calculations revealed the influence of the mutations on the affinity of glyphosate in the PEP-binding site. The amino acid residues of the EPSPS protein in both species involved in glyphosate resistance were elucidated as well as other residues that could be useful for protein engineering. In addition, during MD simulations, we identified conformational changes in glyphosate when complexed with resistant EPSPS, related to loss of herbicide activity and binding affinity. Our computational findings are consistent with previous experimental results and clarify the inhibitory activity of glyphosate as well as the structural target-site resistance of EPSPS against glyphosate.

Fig. 1. Location of analyzed mutations in the structures of Conyza sumatrensis 5-enolpyruvyl shikimate-3-phosphate synthase (CsEPSPS mutant, P106T, panel (A)) and Eleusine indica EPSPS (EiEPSPS double mutant, T102I/P106S, panel (B)). In the active site are the substrate shikimate-3-phosphate identified in purple (S3P) and the inhibitor glyphosate in pink. The 2D structures of S3P and glyphosate are shown in panels (C) and (D), respectively.

Fig. 2. Root-mean-square deviation (RMSD) plots of Conyza sumatrensis 5-enolpyruvyl shikimate-3-phosphate synthase (CsEPSPS) and Eleusine indica EPSPS (EiEPSPS) variants complexed with glyphosate and shikimate-3-phosphate (S3P) obtained over 100 ns of molecular dynamics simulation. (A) CsEPSPS sensitive (orange) and resistant (purple) structures. (B) EiEPSPs sensitive (red) and resistant (blue) structures. The backbone atoms C, N, and O were used in this analysis.

Fig. 3. Average values of the dihedral angles assumed by glyphosate when complexed with resistant and sensitive Conyza sumatrensis 5-enolpyruvyl shikimate-3-phosphate synthase (CsEPSPS) and Eleusine indica EPSPS (EiEPSPS) structures. The dihedral angle values were obtained over 100 ns of molecular dynamics simulation for sensitive and resistant variants of both species.

Fig. 4. Close-up view of the glyphosate (GPJ) binding site of (A and C) wild-type and (B and D) mutants Conyza sumatrensis 5-enolpyruvyl shikimate-3-phosphate synthase (CsEPSPS) and Eleusine indica EPSPS (EiEPSPS). The mutated residues are highlighted in red.

Fig. 5. Pairwise decomposition of residue interactions energies of (A) Conyza sumatrensis 5-enolpyruvyl shikimate-3-phosphate synthase (CsEPSPS) and (B) Eleusine indica EPSPS (EiEPSPS) mutant (resistant, blue) and wild-type structures (sensitive, red).

Fig. 6. (A) Glyphosate (GPJ) complexed with EPSPS binding site interacting with residue Arg362 during the first 30 ns of molecular dynamics simulation. (B) GPJ assuming the condensed conformation in the last 70 ns of the molecular dynamics trajectory.