Interaction of Phosphonate Analogues of the Tetrahedral Reaction Intermediate with 5-Enolpyruvylshikimate-3-phosphate Synthase in Atomic Detail^,

Abstract

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway and is the target of the broad-spectrum herbicide glyphosate. Since the functionality of the shikimate pathway is vital not only for plants but also for microorganisms, EPSPS is considered a prospective target for the development of novel antibiotics. We have kinetically analyzed and determined the crystal structures of Escherichia coli EPSPS inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate. Both diastereomers are competitive inhibitors with respect to the substrates of the EPSPS reaction, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). Remarkably, the (S)-phosphonate (K(iS3P) = 750 nM), whose configuration corresponds to that of the genuine tetrahedral intermediate, is a much weaker inhibitor than the (R)-phosphonate analogue (K(iS3P) = 16 nM). The crystal structures of EPSPS liganded with the (S)- and (R)-phosphonates, at 1.5 and 1.9 A resolution, respectively, revealed that binding of the (R)-phosphonate induces conformational changes of the strictly conserved residues Arg124 and Glu341 within the active site. This appears to give rise to substantial structural alterations in the amino-terminal globular domain of the enzyme. By contrast, binding of the (S)-phosphonate renders the enzyme structure unchanged. Thus, EPSPS may facilitate the tight binding of structurally diverse ligands through conformational flexibility. Molecular docking calculations did not explain why the (R)-phosphonate is the better inhibitor. Therefore, we propose that the structural events during the open-closed transition of EPSPS are altered as a result of inhibitor action.

Characterization of a new type of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericola variabilis

“…EPSPS was identified as the target of the mostly used herbicide glyphosate [4]. Glyphosate mimics the carbocation state of PEP and binds to the binding site of PEP, forming a dead end EPSPS-S3P-glyphosate ternary complex [5][6][7] and thus blocking the formation of EPSP.…”

Molecular basis for the herbicide resistance of Roundup Ready crops

“…Several of our attempts to identify the cation binding site(s) by cocrystallization of the unliganded or S3P-liganded states of the enzyme with the more electron-dense Rb ϩ ion failed. It is conceivable, however, that monovalent cations may bind transiently to the enzyme, acting as chaperones in restructuring the loop, enabling CP4 EPSP synthase to interact more efficiently with PEP during a catalytic cycle (20). Once the loop has adopted a regular structure, the cation-binding site(s) may be lost.…”

Differential Inhibition of Class I and Class II 5-Enolpyruvylshikimate-3-phosphate Synthases by Tetrahedral Reaction Intermediate Analogues,

“…Crystallographic and chemical studies using E. coli EPSPS demonstrated the 2-(S)-configuration of the TI (20,21). The mode of action of glyphosate on EPSPS is well understood (22,23), but intensive efforts to find a molecule better than glyphosate at inhibiting class I EPSPS have largely failed; only a few analogues of the TI have been identified as more potent inhibitors (11,24,25). The molecular modes of action of the (R)-and (S)-phosphonate TI analogues (RP-TI and SP-TI, respectively) on E. coli EPSPS were determined recently by X-ray crystallography and indicated that this enzyme may facilitate the tight binding of such inhibitors through conformational flexibility (25).…”