Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase
1990; American Chemical Society; Volume: 29; Issue: 6 Linguagem: Inglês
10.1021/bi00458a013
ISSN1943-295X
AutoresFrank Rusnak, Jun Liu, Nina Quinn, Glenn A. Berchtold, Christopher T. Walsh,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe 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.
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