Protein Science, Vol 1, Issue 4 549-556, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Characterization of EntF as a serine-activating enzyme

J. REICHERT, M. SAKAITANI and C. T. WALSH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

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-dihydroxybenzoylserine derivatives and exhibits L-serine-dependent ATP[(32)P] pyrophosphate exchange activity with a K(m) 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 enzymebound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate.
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