Computational Study on the Reaction Mechanism of 5-Enolpyruvylshikimate-3-phosphate Synthase from Nicotiana Tabacum.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the conversion of 5-enolpyruvate (PEP) and shikimic acid phosphate (S3P) to 5-enolpyruvylshikimic acid-3-phosphate (EPSP), releasing inorganic phosphate. This reaction is the sixth step of the shikimate pathway, which is a metabolic pathway used by microorganisms and plants for the biosynthesis of aromatic amino acids and folates but not in mammals. In the present study, the detailed reaction mechanism of EPSPS from Nicotiana tabacum (NtEPSPS) is revealed by quantum chemical calculations with the cluster approach. The reaction is proposed to involve the formation of a carbocation intermediate, the formation of a tetrahedral intermediate, the C-O bond cleavage and the re-formation of C=C bond. All four steps are concerted processes involving proton transfer events. The calculations suggest a step-wise mechanism for the formation of the tetrahedral intermediate by the proton transfer from the hydroxyl group of S3P to Asp331 and the nucleophilic attack of hydroxyl group on the carbocation, which is consistent with the proposal in literature. The energy profile for the entire reaction is presented, showing that C-O bond cleavage of the tetrahedral intermediate, releasing phosphate, is the rate-limiting step. The interaction between the Glu359 residue and the phosphate group is significant in stabilizing the phosphate.