title: Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.
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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: F
authors.initials: F
authors.lastname: Rusnak
authors.forename: M
authors.initials: M
authors.lastname: Sakaitani
authors.forename: D
authors.initials: D
authors.lastname: Drueckhammer
authors.forename: J
authors.initials: J
authors.lastname: Reichert
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000032:analysis; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D004758:Enterobactin; D010204:Pantetheine; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; C035180:tyrocidine synthetase; C026269:2,3-dihydroxybenzoate - serine ligase; C003129:4'-phosphopantetheine