Ligand Geometry of the Ternary Complex of 5-Enolpyruvylshikimate-3-phosphate Synthase from Rotational-Echo Double-Resonance NMR

Abstract

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

“…2a). Distances between glyphosate, S3P, and surrounding protein side chain atoms are in good agreement with those predicted from solid-state NMR data (25,26). The 5-hydroxyl group of S3P is hydrogen bonded to the nitrogen atom of glyphosate (Figs.…”

Propagating structure of Alzheimer’s β-amyloid (10–35) is parallel β-sheet with residues in exact register

“…These methods have allowed the measurement of internuclear distances between specific labels to an accuracy of Ϯ0.2 Å (42,(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65). Until now, solid-state NMR had not been used successfully to determine the structures of complex, self-associating proteins such as the amyloids.…”

De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy

“…In contrast, high-resolution solid-state NMR (SSNMR) methods have no inherent molecular weight limit, and have for many years been used to determine details of molecular structure for high molecular weight systems. For example, specific structural features of intact membrane proteins such as bacteriorhodopsin (effective molecular weight Ϸ85,000) (7,8) and large enzyme complexes such as 5-enolpyruvylshikimate-3-phosphate synthase (46,000) (9) and tryptophan synthase (143,000) (10) have been reported. SSNMR methods have also been used to examine surface-bound peptides (11) and to determine a low-resolution structure [1.9-Å backbone rootmean-square deviation (rmsd)] of an insoluble peptide fragment from ␤-amyloid (12) under experimental conditions inaccessible to both solution-state NMR and crystallography.…”