Overexpression, purification and characterization of isochorismate synthase (EntC), the first enzyme involved in the biosynthesis of enterobactin from chorismate

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Abstract
Isochorismate synthase (EC 5.4.99.6), the entC gene product of Escherichia coli, catalyzes ther conversion of chorismate to isochorismate, the first step in the biosynthesis of the powerful iron-clelating agent enterobactin. A sequence-specific deletion method had been used to construct and EntC overproducer, which allows for the purification and characterization of the E. coli isochorismate synthase for the first time. The N-terminal sequence and the subunit molecular weight (43 000) of the polypeptide derived from SDS-polyacrylamide gel electrophoresis agree with those deduced from DNA sequence data. The enzyme is an active monomer with a native molecular weight of 42 000. It was shown that EnTC alone is fully capable of catalyzing the interconversion of chorismate and isochorismate in both directions ans the associated activity is not affected by EntA of the same biosynthetic pathway as has recently been speculated [Elkins, M.F., ''Bacteriol. 171, 791; Ozenberger, B.A., Brickman, T.J., and McIntosh, M.A., (1989) J. Bacteriol. 171, 775]. The kinetic constants were determined with Km 14 .mu.M and Kcat = 173 min-1 for chorismate in the forward direction and Km = 5 .mu.M and = 108 min-1 for isochorismate in the backward direction. The equilibrium constant for the reaction derived from the kinetic data is 0.56 with the equilibrium lying toward the side of chorismate, corresponding to a free energy difference of 0.36 kcal/mol between chorismate and isochorismate. The equilibrium constant was also determined independently by NMR experiments, and it was in agreement with that obtained from kinetic analysis. An H218O labeling experiment established that the incoming hydroxyl group during the reaction was from water rather than by intramolecular transfer from substrate.

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