Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase.
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.
Rusnak
F
F
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Liu
J
J
Quinn
N
N
Berchtold
G A
GA
Walsh
C T
CT
D001483:Base Sequence; D003001:Cloning, Molecular; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012694:Serine / Q000031:analogs & derivatives*; D013379:Substrate Specificity
D004758:Enterobactin; D012694:Serine; D006867:Hydrolases; C064012:isochorismatase