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‐dihydroxybenzoylserine derivatives and exhibits l‐serine‐dependent ATP[³²P] 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 enzyme‐bound intermediate, which has been shown by gel filtration analysis to be (l‐serine)adenylate.