The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.

Isochorismate synthase (EC 5.4.99.6), the entC gene product of Escherichia coli, catalyzes the conversion of chorismate to isochorismate, the first step in the biosynthesis of the powerful iron-chelating agent enterobactin. A sequence-specific deletion method has been used to construct an 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 and the associated activity is not affected by EntA of the same biosynthetic pathway as has recently been speculated [Elkins, M. F., & Earhart, C. F. (1988) FEMS Microbiol. Lett. 56, 35; Liu, J., Duncan, K., & Walsh, C.T. (1989) J. Bacteriol. 171, 791; Ozenberger, B. A., Brickman, T.J., & McIntosh, M. A. (1989) J. Bacteriol. 171, 775]. The kinetic constants were determined with Km = 14 microM and kcat = 173 min-1 for chorismate in the forward direction and Km = 5 microM and kcat = 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzyme 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (2,3-diDHB dehydrogenase, hereafter Ent A), the product of the enterobactin biosynthetic gene entA, catalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic product 2,3-dihydroxybenzoate (2,3-DHB). The catechol 2,3-DHB is one of the key siderophore units of enterobactin, a potent iron chelator secreted by Escherichia coli. To probe the reaction mechanism of this oxidation, a variety of 2,3-diDHB analogues were synthesized and tested as substrates. Specifically, we set out to elucidate both the regio- and stereospecificity of alcohol oxidation as well as the stereochemistry of NAD+ reduction. Of those analogues tested, only those with a C3-hydroxyl group (but not a C2-hydroxyl group) were oxidized to the corresponding ketone products. Reversibility of the Ent A catalyzed reaction was demonstrated with the corresponding NADH-dependent reduction of 3-ketocyclohexane- and cyclohexene-1-carboxylates but not the 2-keto compounds. These results establish that Ent A functions as an alcohol dehydrogenase to specifically oxidize the C3-hydroxyl group of 2,3-diDHB to produce the corresponding 2-hydroxy-3-oxo-4,6-cyclohexadiene-1-carboxylate (Scheme II) as a transient species that undergoes rapid aromatization to give 2,3-DHB. Stereospecificity of the C3 allylic alcohol group oxidation was confirmed to be 3R in a 1R,3R dihydro substrate, 3, and hydride transfer occurs to the si face of enzyme-bound NAD+.