TY - JOUR
T1 - Assembly line enzymology by multimodular nonribosomal peptide synthetases
T2 - The thioesterase domain of E. coil EntF catalyzes both elongation and cyclolactonization
AU - Shaw-Reid, Cathryn A.
AU - Kelleher, Neil L.
AU - Losey, Heather C.
AU - Gehring, Amy M.
AU - Berg, Christian
AU - Walsh, Christopher T.
N1 - Funding Information:
This work was supported in part by National Institutes of Health (NIH) grant GM20011 (C,T.W.). C,A,S,R. is a National Science Foundation Minority Postdoctoral Research Fellow (DBI-9707847) and N,K. is supported by an NIH Postdoctoral Fellowship (F32 AI 10087-02). A.M.G, was a Howard Hughes Medical Institute Predocteral Fellow and H.C,L is a National Science Foundation Graduate Fellow,
PY - 1999/6
Y1 - 1999/6
N2 - Background: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coil 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 Ser1 138→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 TE 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.
AB - Background: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coil 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 Ser1 138→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 TE 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.
KW - Electrospray ionization
KW - Enterobactin
KW - Fourier transform mass spectrometry
KW - Siderophore
KW - Thioesterase
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U2 - 10.1016/S1074-5521(99)80050-7
DO - 10.1016/S1074-5521(99)80050-7
M3 - Article
C2 - 10375542
AN - SCOPUS:0033150199
SN - 2451-9448
VL - 6
SP - 385
EP - 400
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 6
ER -