Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.
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.
Rusnak
F
F
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Sakaitani
M
M
Drueckhammer
D
D
Reichert
J
J
Walsh
C T
CT
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
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