Publications

• Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate.

  Gehring A.M., Bradley K.A., Walsh C.T.

  In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues. We show that EntB, previously ... >> More

  In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues. We show that EntB, previously described as the isochorismate lyase required for production of 2,3-DHB, is a bifunctional protein that also serves as an aryl carrier protein (ArCP) with a role in enterobactin assembly. EntB is phosphopantetheinylated near the C terminus in a reaction catalyzed by EntD with a kcat of 5 min-1 and a Km for apo-EntB of 6.5 microM. This holo-EntB is then acylated with 2,3-DHB in a reaction catalyzed by EntE, previously described as the 2,3-DHB-AMP ligase, with a kcat of 100 min-1 and a Km of <<1 microM for holo-EntB. The N-terminal 187 amino acids of EntB (isochorismate lyase domain) are not needed for reaction of EntB with either EntD or EntE as demonstrated by the equivalent catalytic efficiencies of the full-length EntB (residues 1-285) and the C-terminal EntB ArCP domain (residues 188-285) as substrates for both EntD and EntE. << Less

  Biochemistry 36:8495-8503(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF.

  Gehring A.M., Mori I., Walsh C.T.

  The siderophore molecule enterobactin, a cyclic trimeric lactone of N-(2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherichia coli in response to iron starvation. Here we report the first reconstitution of enterobactin synthetase activity from pure protein components: holo-EntB, E ... >> More

  The siderophore molecule enterobactin, a cyclic trimeric lactone of N-(2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherichia coli in response to iron starvation. Here we report the first reconstitution of enterobactin synthetase activity from pure protein components: holo-EntB, EntE, and holo-EntF. Holo-EntB and holo-EntF were obtained by pretreatment of apo-EntB and apo-EntF with coenzyme A and EntD, thereby eliminating the requirement for EntD in the enterobactin synthetase. 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 EntE, as substrates with a turnover rate of 120-140 min-1. There is no evidence for a stable complex of the enterobactin synthetase components. Mutation of holo-EntF in the thioesterase domain at the putative active site serine residue (Ser1138 to Ala) eliminated enterobactin synthetase activity; however, the mutant holo-EntF retained the ability to adenylate serine and to autoacylate itself by thioester formation between serine and its attached phosphopantetheine cofactor. The mutant holo-EntF also appeared to slowly release N-(2, 3-dihydroxybenzoyl)serine. << Less

  Biochemistry 37:2648-2659(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.