Characterization of EntF as a serine-activating enzyme.
EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF- MK1 strain in the synthesis of 2,3-dihydroxybenzoyl serine derivatives and exhibits L-serine-dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min-1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min-1. These results suggested the presence of an enzyme-bound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate.
Reichert
J
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Sakaitani
M
M
Walsh
C T
CT
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D011756:Diphosphates / Q000378:metabolism; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism*
D011756:Diphosphates; D011994:Recombinant Proteins; D004758:Enterobactin; D012694:Serine; C031920:2,3-dihydroxybenzoylserine; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C026269:2,3-dihydroxybenzoate - serine ligase