Title : Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF - Gehring_1998_Biochemistry_37_2648 |
Author(s) : Gehring AM , Mori I , Walsh CT |
Ref : Biochemistry , 37 :2648 , 1998 |
Abstract :
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. |
PubMedSearch : Gehring_1998_Biochemistry_37_2648 |
PubMedID: 9485415 |
Gene_locus related to this paper: ecoli-entf |
Gene_locus | ecoli-entf |
Gehring AM, Mori I, Walsh CT (1998)
Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF
Biochemistry
37 :2648
Gehring AM, Mori I, Walsh CT (1998)
Biochemistry
37 :2648