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Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization


Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-β-keto intermediates1,2,3. How poly-β-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently4. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 Å crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B1 in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct ‘double hot dog’ (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.

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Figure 1: Biosynthesis of norsolorinic acid anthrone (1) by PksA.
Figure 2: The crystal structure of the PT domain from PksA.
Figure 3: PT structures with bound palmitate or substrate analogue HC8.
Figure 4: Proposed mechanism of cyclizations catalysed by the PT domain.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of palmitate-bound and HC8-bound PT have been deposited in the Protein Data Bank under accession code 3HRQ and 3HRR.


  1. Staunton, J. & Weissman, K. J. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 18, 380–416 (2001)

    Article  CAS  Google Scholar 

  2. Thomas, R. A biosynthetic classification of fungal and Streptomycete fused-ring aromatic polyketides. ChemBioChem 2, 612–627 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  5. Townsend, C. A. & Minto, R. E. in Comprehensive Natural Products 443–471 (Elsevier, 1999)

    Book  Google Scholar 

  6. Udwary, D. W., Merski, M. & Townsend, C. A. Method for prediction of the locations of linker regions within large multifunctional proteins, and application to a type I polyketide synthase. J. Mol. Biol. 323, 585–598 (2002)

    Article  CAS  Google Scholar 

  7. Crawford, J. M., Dancy, B. C. R., 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  ADS  CAS  Google Scholar 

  8. Dillon, S. C. & Bateman, A. The Hotdog fold: wrapping up a superfamily of thioesterases and dehydratases. BMC Bioinformatics 5, 109 (2004)

    Article  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  10. Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 384, 941–953 (2008)

    Article  CAS  Google Scholar 

  11. Koski, K. M., Haapalainen, A. M., Hiltunen, J. K. & Glumoff, T. Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2. J. Mol. Biol. 345, 1157–1169 (2005)

    Article  Google Scholar 

  12. Kimber, M. S. et al. The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa . J. Biol. Chem. 279, 52593–52602 (2004)

    Article  CAS  Google Scholar 

  13. Hibbert, F. & Emsley, J. Hydrogen bonding and chemical reactivity. Adv. Phys. Org. Chem. 26, 255–379 (1991)

    Google Scholar 

  14. Mills, S. G. & Beak, P. Solvent effects on keto-enol equilibria: tests of quantitative models. J. Org. Chem. 50, 1216–1224 (1985)

    Article  CAS  Google Scholar 

  15. Jordan, D. B., Zheng, Y. J., Lockett, B. A. & Basarab, G. S. Stereochemistry of the enolization of scytalone by scytalone dehydratase. Biochemistry 39, 2276–2282 (2000)

    Article  CAS  Google Scholar 

  16. Bahnson, B. J., Anderson, V. E. & Petsko, G. A. Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion. Biochemistry 41, 2621–2629 (2002)

    Article  CAS  Google Scholar 

  17. Sachdeva, S. et al. Separate entrance and exit portals for ligand traffic in Mycobacterium tuberculosis FabH. Chem. Biol. 15, 402–412 (2008)

    Article  CAS  Google Scholar 

  18. Kroken, S., Glass, N. L., Taylor, J. W., Yoder, O. C. & Turgeon, B. G. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic Ascomycetes . Proc. Natl Acad. Sci. USA 100, 15670–15675 (2003)

    Article  ADS  CAS  Google Scholar 

  19. Ma, S. M. et al. Redirecting the cyclization steps of fungal polyketide synthase. J. Am. Chem. Soc. 130, 38–39 (2008)

    Article  CAS  Google Scholar 

  20. Ames, B. D. et al. Crystal structure and functional analysis of tetracenomycin ARO/CYC: implications for cyclization specificity of aromatic polyketides. Proc. Natl Acad. Sci. USA 105, 5349–5354 (2008)

    Article  ADS  Google Scholar 

  21. Verdonk, M. L., Cole, J. C., Hartshorn, M. J., Murray, C. W. & Taylor, R. D. Improved protein-ligand docking using GOLD. Proteins 52, 609–623 (2003)

    Article  CAS  Google Scholar 

  22. Gabb, H. A., Jackson, R. M. & Sternberg, M. J. Modelling protein docking using shape complementarity, electrostatics and biochemical information. J. Mol. Biol. 272, 106–120 (1997)

    Article  CAS  Google Scholar 

  23. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    Article  ADS  CAS  Google Scholar 

  24. Terwilliger, T. C. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  28. Schüttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004)

    Article  Google Scholar 

  29. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  30. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989)

    Article  CAS  Google Scholar 

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We thank T. M. Harris for his gift of HC8. The work at Johns Hopkins was supported by the US National Institutes of Health grant ES001670 awarded to C.A.T. S.-C.T. is supported by the Pew Foundation. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231. J.M.C. is currently a fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2002-09, Harvard Medical School).

Author Contributions J.M.C. carried out biochemical experiments and provided recombinant proteins. A.L.V. carried out all mutational studies. T.P.K. assisted by O.K.-B. determined the PT X-ray crystal structures. E.A.H. prepared substrate and intermediate analogues for co-crystallization experiments. J.W.L. and T.P.K. conducted in silico docking studies. J.M.C., T.P.K. and J.W.L. analysed data and contributed to the writing of the paper, and J.W.L. refined the proposed PT mechanism. S.-C.T. and C.A.T. directed the research, provided funding and edited the final manuscript.

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Correspondence to Shiou-Chuan Tsai or Craig A. Townsend.

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This file contains a Supplementary Discussion, Supplementary Methods, Supplementary Tables S1-S3, Supplementary Figures S1-S6 with Legends and Supplementary References. (PDF 6194 kb)

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Crawford, J., Korman, T., Labonte, J. et al. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization. Nature 461, 1139–1143 (2009).

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