Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1-2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.
Bibliographical noteFunding Information:
This work was supported by grants (GM116957 and GM136235 to A.M.G.) from the National Institutes of Health. We thank G. Wright (McMaster University) for overexpression clones of the E. coli enzymes pantothenate kinase (PanK), phosphopantetheine adenyltransferase (PPAT), and dephosphocoenzyme A kinase (DPCK).
Copyright © 2020 American Chemical Society.
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural