Overexpression, purification, and characterization of isochorismate synthase (EntC), the first enzyme involved in the biosynthesis of enterobactin from chorismate.
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)
Liu
J
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Quinn
N
N
Berchtold
G A
GA
Walsh
C T
CT
D001483:Base Sequence; D002827:Chorismic Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism*; D004758:Enterobactin / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012694:Serine / Q000031:analogs & derivatives*; D013057:Spectrum Analysis; D013816:Thermodynamics
D003509:Cyclohexanecarboxylic Acids; D004758:Enterobactin; D012694:Serine; D007535:Isomerases; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D002827:Chorismic Acid; D010100:Oxygen