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Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Nature volume 454, pages 907–911 (2008)Cite this article

Abstract

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

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Figure 1: Assembly line of the surfactin non-ribosomal peptide synthetase.
Figure 2: Mean structure of SrfTEII.
Figure 3: Complex structure of SrfTEII and the TycC3 T domain.
Figure 4: Size limitation in substrate recognition by SrfTEII.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates have been deposited in the Protein Data Bank under accession number 2RON (structure of SrfTEII) and 2K2Q (complex structure of SrfTEII and H state TycC3–PCP).

References

  1. Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Kopp, F. & Marahiel, M. A. Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Nat. Prod. Rep. 24, 735–749 (2007).

    Article  CAS  Google Scholar 

  3. 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)

    Article  CAS  Google Scholar 

  4. Kim, Y. & Prestegard, J. H. A dynamic model for the structure of acyl carrier protein in solution. Biochemistry 28, 8792–8797 (1989)

    Article  CAS  Google Scholar 

  5. Koglin, A. et al. Conformational switches modulate protein interactions in peptide antibiotic synthetases. Science 312, 273–276 (2006)

    Article  ADS  CAS  Google Scholar 

  6. 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)

    Article  ADS  CAS  Google Scholar 

  7. 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)

    Article  CAS  Google Scholar 

  8. Johnson, M. A., Peti, W., Herrmann, T., Wilson, I. A. & Wüthrich, 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)

    Article  CAS  Google Scholar 

  9. 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)

    Article  CAS  Google Scholar 

  10. Schwarzer, D., Mootz, H. D., Linne, U. & Marahiel, M. A. Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases. Proc. Natl Acad. Sci. USA 99, 14083–14088 (2002).

    Article  ADS  CAS  Google Scholar 

  11. Linne, U., Schwarzer, D., Schroeder, G. N. & Marahiel, M. A. Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis. Eur. J. Biochem. 271, 1536–1545 (2004).

    Article  CAS  Google Scholar 

  12. Yeh, E., Kohli, R. M., Bruner, S. D. & Walsh, C. T. Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated. ChemBioChem 5, 1290–1293 (2004).

    Article  CAS  Google Scholar 

  13. Conti, E., Stachelhaus, T., Marahiel, M. A. & Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997)

    Article  CAS  Google Scholar 

  14. 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)

    Article  CAS  Google Scholar 

  15. Steller, S. et al. Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein. Biochemistry 43, 11331–11343 (2004)

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Schneider, A. & Marahiel, M. A. Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis . Arch. Microbiol. 169, 404–410 (1998)

    Article  CAS  Google Scholar 

  18. Zuiderweg, E. R. P. Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41, 1–7 (2002)

    Article  CAS  Google Scholar 

  19. Frueh, D. P. et al. Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase. Nature 10.1038/nature07162 (this issue)

  20. 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)

    Article  CAS  Google Scholar 

  21. 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).

    Article  CAS  Google Scholar 

  22. Lai, J. R., Koglin, A. & Walsh, C. T. Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis. Biochemistry 45, 14869–14879 (2006).

    Article  CAS  Google Scholar 

  23. Schwarzer, D., Mootz, H. D., Linne, U. & Marahiel, M. A. Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases. Proc. Natl Acad. Sci. USA 99, 14083–14088 (2002)

    Article  ADS  CAS  Google Scholar 

  24. Jenni, S. et al. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science 316, 254–261 (2007)

    Article  ADS  CAS  Google Scholar 

  25. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T 2 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)

    Article  ADS  CAS  Google Scholar 

  26. 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)

    Article  CAS  Google Scholar 

  27. Ferentz, A. E. & Wagner, G. NMR spectroscopy: a multifaceted approach to macromolecular structure. Q. Rev. Biophys. 33, 29–65 (2000)

    Article  CAS  Google Scholar 

  28. Güntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997)

    Article  Google Scholar 

  29. 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)

    Article  CAS  Google Scholar 

  30. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  31. 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)

    Article  CAS  Google Scholar 

  32. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl Acad. Sci. USA 95, 13585–13590 (1998)

    Article  ADS  CAS  Google Scholar 

  33. 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)

    ADS  CAS  Google Scholar 

  34. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004)

    PubMed  Google Scholar 

  35. 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)

    Article  CAS  Google Scholar 

  36. Liang, J., Edelsbrunner, H. & Woodward, C. Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design. Protein Sci. 7, 1884–1897 (1998)

    Article  CAS  Google Scholar 

  37. Dundas, J. et al. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated resiudes. Nucleic Acids Res. 34, W116–W118 (2006)

    Article  CAS  Google Scholar 

  38. Cobas, C. F. & Sardina, J. Nuclear magnetic resonance data processing. MestRe-C: A software package for desktop computers. Concepts Magn. Reson. A 19, 80–96 (2003)

    Article  Google Scholar 

  39. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  40. Belshaw, P. J., Walsh, C. T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999)

    Article  ADS  CAS  Google Scholar 

  41. Sieber, S. A., Walsh, C. T. & Marahiel, M. A. Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains. J. Am. Chem. Soc. 125, 10862–10866 (2003)

    Article  CAS  Google Scholar 

  42. Meier, J. L., Mercer, A. C., Rivera, H. & Burkart, M. D. Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification. J. Am. Chem. Soc. 128, 12174–12184 (2006)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Strieker for editing the manuscript and B. Schaefer for her help and support in sample preparation. We thank Chi Scientific Inc. for the fast and high quality supply of the substrate peptides. The research was funded by the research grant BE-19/11 of the Deutsche Forschungsgemeinschaft (F.B. and M.A.M.); an enclosed fellowship (A.K.); the Centre for Biomolecular Magnetic Resonance at the University Frankfurt; the Cluster of Excellence Frankfurt (Macromolecular Complexes); and the Volkswagen Foundation (P.G.). A.K. thanks the Human Frontier Science Program Organization for a long-term fellowship awarded in April 2007.

Author Contributions A.K., V.D., F.B. and M.A.M. designed the experiments and defined the research for the SrfTEII. A.K., V.D. and C.T.W. wrote the manuscript and designed the experiments for the complex structure. A.K., F.B. and F.L. conducted the research including protein expression, data acquisition, resonance assignment and structure calculation. V.R.R. helped with resonance assignment and structure calculation of the SrfTEII. P.G. provided the volumes of the cavities of SrfTEI and of the complex SrfTEII–TycC3 T domain and supported the complex structure calculation. D.P.F. and G.W. recorded the NMR spectra of the protein complex. A.K. calculated the structure of the isolated SrfTEII, the structure of the protein complex (SrfTEII and TycC3–PCP) and performed all titration experiments. M.A.M. and M.R.M. provided the expression vectors for SrfTEII and the TycC3 T domain. M.A.M. provided the purification protocol and biochemical characterization of the SrfTEII. E.R.S. provided the non-hydrolysable CoA derivate and synthesized the peptide and amino acid modified substrate analogues.

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Authors and Affiliations

  1. Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Macromolecular Complexes (CEF), J.W.-Goethe University, 60438 Frankfurt am Main, Germany

    Alexander Koglin, Frank Löhr, Frank Bernhard, Vladimir V. Rogov, Peter Güntert & Volker Dötsch

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA,

    Alexander Koglin, Dominique P. Frueh, Eric R. Strieter, Gerhard Wagner & Christopher T. Walsh

  3. Institute of Protein Research, 142290 Pushchino, Russia

    Vladimir V. Rogov

  4. Department of Chemistry/Biochemistry, Philipps University, 35032 Marburg, Germany

    Mohammad R. Mofid & Mohamed A. Marahiel

  5. Frankfurt Institute for Advanced Studies, 60438 Frankfurt am Main, Germany

    Peter Güntert

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Correspondence to Christopher T. Walsh, Mohamed A. Marahiel or Volker Dötsch.

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Koglin, A., Löhr, F., Bernhard, F. et al. Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454, 907–911 (2008). https://doi.org/10.1038/nature07161

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