Catalytic Residues and an Electrostatic Sandwich That Promote Enolpyruvyl Shikimate 3-Phosphate Synthase (AroA) Catalysis

Abstract

Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.

Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

“…Therefore, we analyzed the possible reasons resulting in the kinetics difference between them. Based on what is known about the crystal structure of EPSPS, many active sites of EPSPS have been largely studied and identified (3,14,18,19). Mutation of these amino acids can result in a significant change in glyphosate tolerance.…”

Functional Characterization of aroA from Rhizobium leguminosarum with Significant Glyphosate Tolerance in Transgenic Arabidopsis

“…1; we found many similarities between aroA R. leguminosarum and aroA CP4 . As is known to us, many active sites of EPSP synthase have been identified and studied [2]. Alterations at Gly100Ala and Pro105Ser in the EPSPS enzymes result in a significant change in glyphosate tolerance [18].…”

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

“…Glyphosate is a reversible inhibitor of EPSPS because it can bind adjacent to S3P in the PEP binding site and mimic an intermediate state of enzyme–substrates complex. In recent years, a large number of active sites of EPSPS have been studied and identified [17], [18], [19], [20]. The glyphosate binding site is dominated by charged residues Lys22, Arg124, and Lys411, which have previously been concerned in PEP binding [21].…”