Structural and Biochemical Insights into Cotton 5-Enolpyruvylshikimate-3-Phosphate Synthase: Toward Inhibitor Design and Glyphosate Resistance

Cotton (<i>Gossypium</i> spp.), a major fiber crop, requires effective weed control to maintain optimal productivity. Glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in aromatic amino acid biosynthesis; however, the structural mechanisms underlying its inhibition and resistance in plants remain poorly understood. Here, we present the 2.2 Å three-dimensional structure of glyphosate-sensitive <i>Gh</i>EPSPS from cotton in its open conformation. A G178A variant showed markedly reduced glyphosate sensitivity, with an IC₅₀ of 22.27 mM and a dissociation constant of 419 μM compared with 16.4 and 2.7 μM for the wild type. MD simulations revealed that G178A disrupts glyphosate binding, whereas the P183L and TIPS variants remain structurally stable and are unlikely to impair catalytic efficiency. High-throughput virtual screening further identified potential herbicidal chemotypes, and isothermal titration calorimetry confirmed that one of the top candidates, CHEMBL1383715, binds <i>Gh</i>EPSPS with a <i>K</i>_d of 34.5 μM. These findings provide structural insights into glyphosate resistance and highlight CHEMBL1383715 as a promising scaffold for next-generation EPSPS-targeting herbicides.