RECONSTITUTION AND CHARACTERIZATION OF THE ESCHERICHIA-COLI ENTEROBACTIN SYNTHETASE FROM ENTB, ENTE, AND ENTF

The siderophore molecule enterobactin, a cyclic trimeric lactone of N- (2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherich ia coli In response to iron starvation. Here we report the first recon stitution of enterobactin synthetase activity from pure protein compon ents: holo-EntB, Ent, and holo-EntF. Holo-EntB and holo-EntF were obta ined by pretreatment of apo-EntB and apo-EntF with coenzyme A and EntD , thereby eliminating the requirement for EntD in the enterobactin syn thetase. The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L- serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by Ent E, as substrates with a turnover rate of 120-140 min(-1). There is no evidence for a stable complex of the enterobactin synthetase component s. Mutation of holo-EntF in the thioesterase domain at the putative ac tive site serine residue (Ser1138 to Ala) eliminated enterobactin synt hetase activity however, the mutant holo-EntF retained the ability to adenylate serine and to autoacylate itself by thioester formation betw een serine and its attached phosphopantetheine cofactor. The mutant ho lo-EntF also appeared to slowly release N-(2,3-dihydroxybenzoyl)serine .