Unraveling the atomic mechanisms underlying glyphosate insensitivity in EPSPS: implications of distal mutations

5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), as an indispensable enzyme in the shikimate pathway, is the specific target of grasser killer glyphosate (GPJ). GPJ is a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. A novel Ls-EPSPS gene variant discovered from Liliaceae, named ELs-EPSPS, includes five distal mutations, E112V, D142N, T351S, D425G, and R496G, endowing high GPJ insensitivity. However, the implicit molecular mechanism of the enhanced tolerance/insensitivity of GPJ in ELs-EPSPS is not fully understood. Herein, we try to interpret the hidden molecular mechanism using computational methods. Computational results reveal the enhanced flexibility of apo EPSPS upon mutations. The enhanced affinity of the initial binding substrate shikimate-3-phosphate (S3P), and the higher probability of second ligands PEP/GPJ entering the pocket are observed in the ELs-EPSPS-S3P system. Docking and MD results further confirmed the decreased GPJ-induced EPSPS inhibition upon mutations. And, the alterations of K98 and R179 side-chain orientations upon mutations are detrimental to GPJ binding at the active site. Additionally, the oscillation of side chain K98, in charge of PEP location, improves the proximity effect for substrates in the dual-substrate systems upon mutations. Our results clarify that the enhanced GPJ tolerance of EPSPS is achieved from decreased competitive inhibition of GPJ at the atomic perspective, and this finding further contributes to the cultivation of EPSPS genes with higher GPJ tolerance/insensitivity and a mighty renovation for developing glyphosate-resistant crops. Communicated by Ramaswamy H. Sarma.