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-peptidy l carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzy me 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 linkag es and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl car rier protein) intermediate, we mutated residues of the proposed catalytic S er-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) d imer (25%) as products. The His1271-->Ala mutant (k(cat) decreased 10,000-f old compared with wild-type EntF) releases only enterobactin, but accumulat es both DHB-Ser-O-TE and (DHB-Ser)(2)-O-TE acyl enzyme intermediates. Elect rospray ionization and Fourier transform mass spectrometry of proteolytic d igests were used to analyze the intermediates. Conclusions: These results establish that the IE domain of EntF is both a c yclotrimerizing lactone synthetase and an elongation catalyst for ester-bon d formation between covalently tethered DHB-Ser moieties, a new function fo r chain-termination TE domains found at the carboxyl termini of multimodula r NRPSs and polyketide synthases.