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

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti-stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6-fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406).