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 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.
Gehring
A M
AM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Mori
I
I
Walsh
C T
CT
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry
D017931:DNA Primers; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D004758:Enterobactin; D012694:Serine; D006867:Hydrolases; C064012:isochorismatase; D008025:Ligases; C066234:enterobactin synthetase; D010453:Peptide Synthases; C026269:2,3-dihydroxybenzoate - serine ligase; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli