Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The structural organization of substrate loading in iterative polyketide synthases

This article has been updated

Abstract

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/–condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Domain organization and catalytic scheme of the cercosporin PKS CTB1.
Fig. 2: Crystal structure and interdomain interactions in CTB1 SAT-KS-MAT.
Fig. 3: Asymmetric cryo-EM structure of CTB1 SAT°-KS-MAT°=ACP2.
Fig. 4: Schematic illustration of suggested modes of conformational coupling in CTB1.

Similar content being viewed by others

Change history

  • 10 April 2018

    In the version of this article originally published, “Supplementary Text and Figures” in the Supplementary Information section incorrectly linked to the Life Sciences Reporting Summary instead of the file containing Supplementary Tables 1–4 and Supplementary Figures 1–12. The error has been corrected in the HTML version of this article.

References

  1. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Edn. Engl. 48, 4688–4716 (2009).

    Article  CAS  Google Scholar 

  2. Herbst, D. A., Jakob, R. P., Zähringer, F. & Maier, T. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases. Nature 531, 533–537 (2016).

    Article  CAS  Google Scholar 

  3. Weissman, K. J. The structural biology of biosynthetic megaenzymes. Nat. Chem. Biol. 11, 660–670 (2015).

    Article  CAS  Google Scholar 

  4. Tang, Y., Kim, C. Y., Mathews, I. I., Cane, D. E. & Khosla, C. The 2.7-angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase. Proc. Natl Acad. Sci. USA 103, 11124–11129 (2006).

    Article  CAS  Google Scholar 

  5. Maier, T., Leibundgut, M. & Ban, N. The crystal structure of a mammalian fatty acid synthase. Science 321, 1315–1322 (2008).

    Article  CAS  Google Scholar 

  6. Crawford, J. M., Vagstad, A. L., Whitworth, K. P., Ehrlich, K. C. & Townsend, C. A. Synthetic strategy of nonreducing iterative polyketide synthases and the origin of the classical “starter-unit effect”. ChemBioChem 9, 1019–1023 (2008).

    Article  CAS  Google Scholar 

  7. Vagstad, A. L. et al. Combinatorial domain swaps provide insights into the rules of fungal polyketide synthase programming and the rational synthesis of non-native aromatic products. Angew. Chem. Int. Edn. Engl. 52, 1718–1721 (2013).

    Article  CAS  Google Scholar 

  8. Foulke-Abel, J. & Townsend, C. A. Demonstration of starter unit interprotein transfer from a fatty acid synthase to a multidomain, nonreducing polyketide synthase. ChemBioChem 13, 1880–1884 (2012).

    Article  CAS  Google Scholar 

  9. Crawford, J. M., Dancy, B. C., Hill, E. A., Udwary, D. W. & Townsend, C. A. Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase. Proc. Natl Acad. Sci. USA 103, 16728–16733 (2006).

    Article  CAS  Google Scholar 

  10. Newman, A. G., Vagstad, A. L., Storm, P. A. & Townsend, C. A. Systematic domain swaps of iterative, nonreducing polyketide synthases provide a mechanistic understanding and rationale for catalytic reprogramming. J. Am. Chem. Soc. 136, 7348–7362 (2014).

    Article  CAS  Google Scholar 

  11. Fujii, I., Watanabe, A., Sankawa, U. & Ebizuka, Y. Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem. Biol. 8, 189–197 (2001).

    Article  CAS  Google Scholar 

  12. Dutta, S. et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014).

    Article  CAS  Google Scholar 

  13. Tang, Y., Chen, A. Y., Kim, C. Y., Cane, D. E. & Khosla, C. Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase. Chem. Biol. 14, 931–943 (2007).

    Article  CAS  Google Scholar 

  14. Whicher, J. R. et al. Cyanobacterial polyketide synthase docking domains: a tool for engineering natural product biosynthesis. Chem. Biol. 20, 1340–1351 (2013).

    Article  CAS  Google Scholar 

  15. Pappenberger, G. et al. Structure of the human fatty acid synthase KS-MAT didomain as a framework for inhibitor design. J. Mol. Biol. 397, 508–519 (2010).

    Article  CAS  Google Scholar 

  16. Vagstad, A. L., Bumpus, S. B., Belecki, K., Kelleher, N. L. & Townsend, C. A. Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity. J. Am. Chem. Soc. 134, 6865–6877 (2012).

    Article  CAS  Google Scholar 

  17. Crawford, J. M. et al. Deconstruction of iterative multidomain polyketide synthase function. Science 320, 243–246 (2008).

    Article  CAS  Google Scholar 

  18. Winter, J. M. et al. Biochemical and structural basis for controlling chemical modularity in fungal polyketide biosynthesis. J. Am. Chem. Soc. 137, 9885–9893 (2015).

    Article  CAS  Google Scholar 

  19. Liu, T., Chiang, Y. M., Somoza, A. D., Oakley, B. R. & Wang, C. C. Engineering of an “unnatural” natural product by swapping polyketide synthase domains in Aspergillus nidulans. J. Am. Chem. Soc. 133, 13314–13316 (2011).

    Article  CAS  Google Scholar 

  20. Bruegger, J. et al. Probing the selectivity and protein•protein interactions of a nonreducing fungal polyketide synthase using mechanism-based crosslinkers. Chem. Biol. 20, 1135–1146 (2013).

    Article  CAS  Google Scholar 

  21. Choquer, M. et al. The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae. Mol. Plant Microbe Interact. 18, 468–476 (2005).

    Article  CAS  Google Scholar 

  22. Chung, K. R., Ehrenshaft, M., Wetzel, D. K. & Daub, M. E. Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae. Mol. Genet. Genomics 270, 103–113 (2003).

    Article  CAS  Google Scholar 

  23. Daub, M. E. & Ehrenshaft, M. The photoactivated cercospora toxin cercosporin: contributions to plant disease and fundamental biology. Annu. Rev. Phytopathol. 38, 461–490 (2000).

    Article  CAS  Google Scholar 

  24. Newman, A. G., Vagstad, A. L., Belecki, K., Scheerer, J. R. & Townsend, C. A. Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function. Chem. Commun. (Camb.) 48, 11772–11774 (2012).

    Article  CAS  Google Scholar 

  25. Nguyen, C. et al. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis. Nature 505, 427–431 (2014).

    Article  CAS  Google Scholar 

  26. Whicher, J. R. et al. Structural rearrangements of a polyketide synthase module during its catalytic cycle. Nature 510, 560–564 (2014).

    Article  CAS  Google Scholar 

  27. Kapur, S. et al. Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation. Proc. Natl Acad. Sci. USA 109, 4110–4115 (2012).

    Article  CAS  Google Scholar 

  28. Wang, F. et al. Structural and functional analysis of the loading acyltransferase from avermectin modular polyketide synthase. ACS Chem. Biol. 10, 1017–1025 (2015).

    Article  CAS  Google Scholar 

  29. Wattana-amorn, P. et al. Solution structure of an acyl carrier protein domain from a fungal type I polyketide synthase. Biochemistry 49, 2186–2193 (2010).

    Article  CAS  Google Scholar 

  30. 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  CAS  Google Scholar 

  31. Changeux, J. P. & Edelstein, S. Conformational selection or induced fit? 50 years of debate resolved. F1000 Biol. Rep. 3, 19 (2011).

    Article  Google Scholar 

  32. Brignole, E. J., Smith, S. & Asturias, F. J. Conformational flexibility of metazoan fatty acid synthase enables catalysis. Nat. Struct. Mol. Biol. 16, 190–197 (2009).

    Article  CAS  Google Scholar 

  33. Maier, T., Jenni, S. & Ban, N. Architecture of mammalian fatty acid synthase at 4.5 A resolution. Science 311, 1258–1262 (2006).

    Article  CAS  Google Scholar 

  34. Zhang, L. et al. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis. Cell Res. 26, 1330–1344 (2016).

    Article  CAS  Google Scholar 

  35. Keatinge-Clay, A. T., Maltby, D. A., Medzihradszky, K. F., Khosla, C. & Stroud, R. M. An antibiotic factory caught in action. Nat. Struct. Mol. Biol. 11, 888–893 (2004).

    Article  CAS  Google Scholar 

  36. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  37. Betancor, L., Fernández, M. J., Weissman, K. J. & Leadlay, P. F. Improved catalytic activity of a purified multienzyme from a modular polyketide synthase after coexpression with Streptomyces chaperonins in Escherichia coli. ChemBioChem 9, 2962–2966 (2008).

    Article  CAS  Google Scholar 

  38. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  39. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, (213–221 (2010).

    Google Scholar 

  40. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  41. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  Google Scholar 

  42. Cowtan, K. Recent developments in classical density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 470–478 (2010).

    Article  CAS  Google Scholar 

  43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  44. Edwards, A. L., Matsui, T., Weiss, T. M. & Khosla, C. Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase. J. Mol. Biol. 426, 2229–2245 (2014).

    Article  CAS  Google Scholar 

  45. Asturias, F. J. et al. Structure and molecular organization of mammalian fatty acid synthase. Nat. Struct. Mol. Biol. 12, 225–232 (2005).

    Article  CAS  Google Scholar 

  46. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  Google Scholar 

  47. McLeod, R. A., Kowal, J., Ringler, P. & Stahlberg, H. Robust image alignment for cryogenic transmission electron microscopy. J. Struct. Biol. 197, 279–293 (2017).

    Article  CAS  Google Scholar 

  48. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  49. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  Google Scholar 

  50. Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

    Article  Google Scholar 

  51. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  Google Scholar 

  52. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  53. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

    Article  CAS  Google Scholar 

  54. Urnavicius, L. et al. The structure of the dynactin complex and its interaction with dynein. Science 347, 1441–1446 (2015).

    Article  CAS  Google Scholar 

  55. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  Google Scholar 

  56. Bai, X. C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

    Article  Google Scholar 

  57. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

    Article  Google Scholar 

  58. 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 

  59. McLeod, R. A., Diogo Righetto, R., Stewart, A. & Stahlberg, H. MRCZ - A file format for cryo-TEM data with fast compression. J. Struct. Biol. 201, 252–257 (2017).

  60. Schwede, T., Kopp, J., Guex, N. & Peitsch, M. C. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003).

    Article  CAS  Google Scholar 

  61. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    Article  CAS  Google Scholar 

  62. Kleywegt, G. J. Validation of protein models from Cα coordinates alone. J. Mol. Biol. 273, 371–376 (1997).

    Article  CAS  Google Scholar 

  63. Kleywegt, G. J. Use of non-crystallographic symmetry in protein structure refinement. Acta Crystallogr. D Biol. Crystallogr. 52, 842–857 (1996).

    Article  CAS  Google Scholar 

  64. Kahraman, A., Malmström, L. & Aebersold, R. Xwalk: computing and visualizing distances in cross-linking experiments. Bioinformatics 27, 2163–2164 (2011).

    Article  CAS  Google Scholar 

  65. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  Google Scholar 

Download references

Acknowledgements

We thank P. Leadlay and L. Betancor for providing plasmid pETcoco-2A-L1SL2. X-ray diffraction data were collected at beamline PXI of the Paul Scherrer Institute, Villigen, Switzerland, and cryo-EM data were collected at the BioEM facility of the University of Basel; we acknowledge excellent support by teams of both facilities. This work was supported by the Swiss National Science Foundation (SNF) project grants 138262, 159696, SNF R’equip grants 145023 and 164074, and the National Institutes of Health (ES001670). D.A.H. acknowledges a fellowship from the Werner-Siemens Foundation.

Author information

Authors and Affiliations

Authors

Contributions

R.P.J. expressed, purified and crystallized CTB1 SAT-KS-MAT. D.A.H., R.P.J., and T.M. solved the crystal structure. D.A.H. performed cryo-EM, data processing, modeling, refinement and analysis of all structural data. C.R.H.-R. optimized and prepared crosslinked CTB1 SAT°-KS-MAT°=ACP2 for structural analysis and performed mutational experiments for structural validation. J.M.K. synthesized the α-bromopropionyl crosslinker. P.A.S. and J.R.A. performed initial exploratory experiments, and J.R.A. prepared the CTB1 SAT°-KS-MAT° construct for crosslinking. C.A.T. and T.M. designed research. The manuscript was written by D.A.H., T.M., C.R.H.-R., and C.A.T.

Corresponding author

Correspondence to Timm Maier.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–4 and Supplementary Figures 1–12

Life Sciences Reporting Summary

Supplementary Note 1:

Synthetic Procedures

Supplementary Video 1

ACP2 binding to the CTB1 loading/condensing region

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Herbst, D.A., Huitt-Roehl, C.R., Jakob, R.P. et al. The structural organization of substrate loading in iterative polyketide synthases. Nat Chem Biol 14, 474–479 (2018). https://doi.org/10.1038/s41589-018-0026-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0026-3

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing