Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine. |
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Authors: | F Rusnak M Sakaitani D Drueckhammer J Reichert C T Walsh |
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Institution: | Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115. |
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Abstract: | The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor. |
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