Assembly line enzymology by multimodular nonribosomal peptide synthetases:: the thioesterase domain of E-coli EntF catalyzes both elongation and cyclolactonization

Background: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. Results: The Ser1138-->Cys mutant (k(cat) decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)(2) dimer (25%) as products. The His1271-->Ala mutant (k(cat) decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)(2)-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. Conclusions: These results establish that the IE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.