Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties1. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations2. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Scientific Reports Open Access 26 March 2019
BMC Microbiology Open Access 13 August 2015
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810 (2004)
Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L. O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007)
Gehring, A. M., Bradley, K. A. & Walsh, C. T. Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate. Biochemistry 36, 8495–8503 (1997)
Gehring, A. M., Mori, I. & Walsh, C. T. Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochemistry 37, 2648–2659 (1998)
Wagner, G. The importance of being floppy. Nature Struct. Biol. 2, 255–257 (1995)
Koglin, A. et al. Conformational switches modulate protein interactions in peptide antibiotic synthetases. Science 312, 273–276 (2006)
Weber, T., Baumgartner, R., Renner, C., Marahiel, M. A. & Holak, T. A. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases. Structure 8, 407–418 (2000)
Findlow, S. C., Winsor, C., Simpson, T. J., Crosby, J. & Crump, M. P. Solution structure and dynamics of oxytetracycline polyketide synthase acyl carrier protein from Streptomyces rimosus . Biochemistry 42, 8423–8433 (2003)
Johnson, M. A., Peti, W., Herrmann, T., Wilson, I. A. & Wuthrich, K. Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC 7120 with a variant phosphopantetheinylation-site sequence. Protein Sci. 15, 1030–1041 (2006)
Zornetzer, G. A., Fox, B. G. & Markley, J. L. Solution structures of spinach acyl carrier protein with decanoate and stearate. Biochemistry 45, 5217–5227 (2006)
Kim, Y. & Prestegard, J. H. A dynamic model for the structure of acyl carrier protein in solution. Biochemistry 28, 8792–8797 (1989)
Bruner, S. D. et al. Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE. Structure 10, 301–310 (2002)
Samel, S. A., Wagner, B., Marahiel, M. A. & Essen, L. O. The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-ribosomal lipopeptide. J. Mol. Biol. 359, 876–889 (2006)
Koglin, A. et al. Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 10.1038/nature07161 (this issue)
Leibundgut, M., Jenni, S., Frick, C. & Ban, N. Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science 316, 288–290 (2007)
Lomakin, I. B., Xiong, Y. & Steitz, T. A. The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together. Cell 129, 319–332 (2007)
Maier, T., Jenni, S. & Ban, N. Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science 311, 1258–1262 (2006)
Jenni, S., Leibundgut, M., Maier, T. & Ban, N. Architecture of a fungal fatty acid synthase at 5 Å resolution. Science 311, 1263–1267 (2006)
Jenni, S. et al. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science 316, 254–261 (2007)
Lambalot, R. H. et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 3, 923–936 (1996)
Lambalot, R. H. & Walsh, C. T. Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J. Biol. Chem. 270, 24658–24661 (1995)
Roche, E. D. & Walsh, C. T. Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. Biochemistry 42, 1334–1344 (2003)
Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wuthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl Acad. Sci. USA 95, 13585–13590 (1998)
Rovnyak, D. et al. Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J. Magn. Reson. 170, 15–21 (2004)
Frueh, D. P. et al. Non-uniformly sampled double-TROSY hNcaNH experiments for NMR sequential assignments of large proteins. J. Am. Chem. Soc. 128, 5757–5763 (2006)
Frueh, D. P., Vosburg, D. A., Walsh, C. T. & Wagner, G. Determination of all NOes in 1H–13C-Me-ILV-U-2H–15N proteins with two time-shared experiments. J. Biomol. NMR 34, 31–40 (2006)
Guntert, P., Mumenthaler, C. & Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997)
Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997)
Tolman, J. R., Flanagan, J. M., Kennedy, M. A. & Prestegard, J. H. Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc. Natl Acad. Sci. USA 92, 9279–9283 (1995)
Gardner, K. H. & Kay, L. E. Production and incorporation of 15N, 13C, 2H (1H-δ1 methyl) isoleucine into proteins for multidimensional NMR studies. J. Am. Chem. Soc. 119, 7599–7600 (1997)
Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E. & Dahlquist, F. W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 177, 44–73 (1989)
Ferentz, A. E. & Wagner, G. NMR spectroscopy: a multifaceted approach to macromolecular structure. Q. Rev. Biophys. 33, 29–65 (2000)
Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999)
Hyberts, S. G. & Wagner, G. IBIS – a tool for automated sequential assignment of protein spectra from triple resonance experiments. J. Biomol. NMR 26, 335–344 (2003)
Pervushin, K., Riek, R., Wider, G. & Wuthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997)
Pervushin, K. & Eletsky, A. A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment. J. Biomol. NMR 25, 147–152 (2003)
Grzesiek, S., Anglister, J. & Bax, A. Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J. Magn. Reson. B. 101, 114–119 (1993)
Bax, A., Clore, M. & Gronenborn, A. M. 1H-1H Correlation via isotropic mixing of 13C magentization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J. Magn. Reson. 88, 425–431 (1990)
Ikura, M., Kay, L. E. & Bax, A. Improved three-dimensional 1H-13C-1H correlation spectroscopy of a 13C-labeled protein using constant-time evolution. J. Biomol. NMR 1, 299–304 (1991)
Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)
Medek, A., Olejniczak, E. T., Meadows, R. P. & Fesik, S. W. An approach for high-throughput structure determination of proteins by NMR spectroscopy. J. Biomol. NMR 18, 229–238 (2000)
Delaglio, F. et al. NMRPipe a Multidimensional Spectra Processing System Based on UNIX Pipes. J. Biomol. NMR 6, 277–293 (1995)
Keller, R. L. J. The Computer Aided Resonance Assignment Tutorial (Cantina, 2004)
Schwede, T., Kopp, J., Guex, N. & Peitsch, M. C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003)
Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)
Nederveen, A. J. et al. RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank. Proteins 59, 662–672 (2005)
Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
We thank M. Sastry and A. Marintchev for advice in sample preparation; Z. Zhou for providing the Ser48Ala plasmid; Z.-Y. Sun for participating in the development of new NMR techniques; and P. Selenko for comments on the manuscript. This work was supported by NIH grants GM47467, EB 002026, and GM066360 and a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund (D.A.V.). A.K. thanks the Human Frontier Science Program for a long-term fellowship awarded in April 2007.
Author Contributions D.P.F., G.W. and C.T.W. designed research and wrote the manuscript. D.P.F. conducted the research including protein expression, resonance assignment and structure calculation. H.A. prepared various samples and helped during the acquisition of NMR experiments. D.A.V. prepared various samples. A.K. helped during the structure calculation and prepared the C domains EntD and Sfp samples. A.E.B. participated in early stages of the project.
About this article
Cite this article
Frueh, D., Arthanari, H., Koglin, A. et al. Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase. Nature 454, 903–906 (2008). https://doi.org/10.1038/nature07162
Scientific Reports (2019)
Journal of Industrial Microbiology and Biotechnology (2019)
The evolution of three siderophore biosynthetic clusters in environmental and host-associating strains of Pantoea
Molecular Genetics and Genomics (2018)
Nature Chemical Biology (2017)