Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Abstract

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC 50 of the fluorinated substrate analogue was 0.5 and 250 M with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) 1 to chorismate (Scheme 1) (1). This enzyme is the seventh enzyme of the shikimate pathway and is present in bacteria, fungi, and plants but not in mammals. It is therefore a potential target for antimicrobial agents and herbicides. The strict requirement for a reduced FMN cofactor (2-4) and the trans-1,4-elimination of the 3-phosphate and the 6R-H (5-7) are both unusual aspects of the chorismate synthase reaction. A number of non-concerted mechanisms have been proposed to account for these properties (1,8,9).Evidence for a non-concerted mechanism includes a secondary tritium kinetic isotope effect at C(3) (10), the slow conversion of (6S)-6-fluoro-EPSP to 6-fluoro-chorismate (11), transient kinetics studies (12), and more recently, a secondary ␤ deuterium kinetic isotope effect at C(4) (13). These studies collectively make the radical (Scheme 1A) and E1 (Scheme 1B) mechanisms involving the initial cleavage of the C(3)-O bond the most likely, and alternative mechanisms where the C(6)-H bond breaks first the least likely (8,14). Evidence for the radical mechanism (Scheme 1A), which provides a role for the reduced flavin in catalysis, comes from the lack of activity of the bifunctional Neurospora crassa enzyme with 5-deaza-FMN (15) and the formation of a flavin semiquinone with the monofunctional Escherichia coli enzyme and the inhibitor (6R)-6-fluoro-EPSP (16).This paper describes new studies with the E. coli enzyme that are strongly supportive of the proposed radical mechanism (Scheme 1A). The product of the reaction with the inhibitor (6R)-6-fluoro-EPSP has been identified, allowing the formation of the stable flavin semiquinone radical in the presence of dithionite to be rationalized. The formation of a flavin semiquinone radical during turnover of (6S)-6-fluoro-EPSP has been observed in the prese...

Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa

“…The blue shift of the flavin fluorescence maximum at 525 nm and the occurrence of an additional peak at 450 nm also suggest a more hydrophobic environment upon binding of EPSP to the binary NcCS⅐FMN complex (6). The hypothesis has been put forward that the hydrophobicity of the FMN binding pocket renders the reduced flavin a better electron donor for its assumed role as a transient electron transfer agent to EPSP to initiate cleavage of the C-O bond (4,5). However, to date no flavin radical species has been observed spectroscopically during EPSP turnover.…”

“…Although the reaction does not involve a net change in redox state, the enzyme has an absolute requirement for reduced FMN that is not consumed during substrate turnover. The role of reduced FMN has been subject to intense mechanistic studies (reviewed in Macheroux et al (3)) that have led to a proposal of a mechanism involving radical chemistry (4,5). Another issue revolving around the requirement for reduced flavin concerns the generation of the reduced cofactor and its sequestration by chorismate synthase.…”

2-Haloacrylate Hydratase, a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

“…The reaction was terminated by the addition of 200 l acetonitrile to the mixture, and the mixture was centrifuged, filtered, diluted with acetonitrile-10 mM ammonium acetate (1:1), and then introduced into the mass spectrometer in the negative-ion mode at 5 l/min. For 18 O incorporation experiments, the reaction was carried out in a standard assay mixture containing 50% (vol/vol) H 2 18 O. The protein concentration was determined according to the method of Bradford, with bovine serum albumin as a standard (3).…”

Comparison of DFT and ab initio QM/MM methods for modelling reaction in chorismate synthase

“…It has been suggested that the reaction involves an unstable intermediate, formed by the initial loss of phosphate, which may be of a radical nature [10], but a cationic mechanism cannot yet be ruled out.…”