Escherichia coli Chorismate Synthase Catalyzes the Conversion of (6S)-6-Fluoro-5-enolpyruvylshikimate-3-phosphate to 6-Fluorochorismate

Abstract

Chorismate synthase (EC 4.6.1.4) is the seventh enzyme of the shikimate pathway (1, 2) and catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate 1 (EPSP) 1 to chorismate 2 (Scheme 1). Chorismate is a common intermediate in the biosynthesis of aromatic metabolites such as aromatic amino acids and folate cofactors. The chorismate synthase reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen with overall anti-stereochemistry (3-5). A concerted elimination appears to be unlikely since studies with model systems (6, 7) and molecular orbital considerations (8, 9) suggest that concerted 1,4-eliminations with syn-stereochemistry are favored. For these reasons, several non-concerted mechanisms have been suggested for the chorismate synthase reaction (10). Proposed mechanisms include an X-group mechanism, involving the nucleophilic attack of the C-1 position of the substrate (5) (Scheme 2d), and a cationic mechanism, involving the step-wise loss of phosphate and a proton (11, 12) (Scheme 2a). An allylic rearrangement of phosphate followed by a 1,2-elimination (13) has been discounted because the intermediate iso-EPSP is a competitive inhibitor rather than a substrate of the Neurospora crassa enzyme (14).Reduced FMN is required for enzyme activity despite there being no overall reduction or oxidation in the conversion of EPSP to chorismate (15)(16)(17)(18)(19). The role of flavin is not yet clear but it appears to be directly involved in catalysis. A transient flavin intermediate has been characterized by uv/visible spectroscopy during single and multiple turnover experiments using the enzyme from Escherichia coli (20,21). It has been suggested that this spectrum is consistent with a charge transfer complex or a C-4a-flavin adduct (20). However, such a perturbation of the reduced flavin spectrum could, at least in part, be due to noncovalent interactions between the substrate and the flavin. The absence of detectable activity of the N. crassa (22) and E. coli (23) enzymes reconstituted with reduced 5-deaza-FMN provides additional evidence that reduced flavin is chemically, and not just structurally, involved in turnover. In the presence of the inhibitor (6R)-6-fluoro-EPSP the E. coli enzyme forms a protein-bound flavin semiquinone, suggesting the possibility of radical intermediates during normal turnover (24). A radical mechanism involving the initial abstraction of a hydrogen atom from the C-6 position of the substrate (11, 22, 25) seems unlikely since there are no obvious candidates for a catalytic center that can accept single electrons, such as transition metal ions (24), particularly given the requirement for fully reduced flavin for activity.

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

“…is slowly but quantitatively converted to 6-fluoro-chorismate by chorismate synthase under steady-state conditions whether the flavoholoenzyme is reduced by dithionite or by photoirradiation before the addition of the substrate analogue (11). We now report that the enzyme forms a transient semiquinone flavin radical during multiple turnover experiments in the presence of dithionite-but not photo-reduced flavin.…”

“…The resulting allylic anion is very likely to eliminate fluoride leading to the formation of chorismate is this case also. The only product detected (Ͼ99%) in steady-state assay mixtures containing dithionite is 6-fluorochorismate (11). Therefore, it would appear that the active sites that undergo this chemistry are blocked with regard to the catalytic formation of chorismate, as is the case with (6R)-6-fluoro-EPSP.…”

“…Anaerobic assay mixtures contained chorismate synthase (between 54 and 245 milliunits), EPSP (50 M), 5-deaza-FMN (between 20 and 50 M), sodium dithionite (1 mM), and MOPS buffer. Reactions were started with the addition of either enzyme or substrate and incubated for between 2.5 and 10 min before HPLC analysis of chorismate using a SAX column as described previously (11,12).…”

“…The transient formation of a neutral reduced flavin is observed during turnover of the normal substrate (17,26,28) and during a single turnover of (6S)-6-fluoro-EPSP (11). On the addition of an 18-fold excess of (6S)-6-fluoro-EPSP to the enzyme-bound monoanionic dithionite-reduced FMN (Fig.…”

“…7, circles). It was estimated that about 90% of the active sites were converted to this form (⑀ 386 ϳ6000 M Ϫ1 cm Ϫ1 (11,12)). Unusually, a little semiquinone radical had also formed within 15 s, which accounted for the remaining 10% of the active sites.…”