PMID:9485415
| Citation |
Gehring, AM, Mori, I and Walsh, CT (1998) Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochemistry 37:2648-59 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi9726584 |
| Keywords |
Base Sequence; Binding Sites/genetics; DNA Primers/genetics; Enterobactin/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Hydrolases/chemistry; Hydrolases/genetics; Hydrolases/isolation & purification; Ligases/chemistry; Ligases/genetics; Ligases/isolation & purification; Multienzyme Complexes/chemistry; Multienzyme Complexes/genetics; Multienzyme Complexes/isolation & purification; Mutagenesis, Site-Directed; Peptide Synthases/chemistry; Peptide Synthases/genetics; Peptide Synthases/isolation & purification; Point Mutation; Protein Conformation; Serine/chemistry |
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