Letter

Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases

Received:
Accepted:
Published online:

Abstract

Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities1,2,3. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line3, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs)3. However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment4,5. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases6. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like7,8 PKSs. It is involved in the biosynthesis of C20–C28 branched-chain fatty acids, which are important virulence factors of mycobacteria9. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

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Change history

  • Corrected online 23 March 2016

    Figure 4b was corrected to include a rotation axis line.

Accessions

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession numbers 5BP1, 5BP2, 5BP3, 5BP4.

References

  1. 1.

    Uncovering the structures of modular polyketide synthases. Nat. Prod. Rep. 32, 436–453 (2015)

  2. 2.

    & Combinatorial biosynthesis of polyketides—a perspective. Curr. Opin. Chem. Biol. 16, 117–123 (2012)

  3. 3.

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

  4. 4.

    et al. Multifactorial control of iteration events in a modular polyketide assembly line. Angew. Chem. Int. Edn Engl . 52, 5285–5289 (2013)

  5. 5.

    , & Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity. Nat. Prod. Rep. 21, 575–593 (2004)

  6. 6.

    , & The crystal structure of a mammalian fatty acid synthase. Science 321, 1315–1322 (2008)

  7. 7.

    , , , & The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J. Biol. Chem. 276, 16833–16839 (2001)

  8. 8.

    & Polyketide versatility in the biosynthesis of complex mycobacterial cell wall lipids. Methods Enzymol. 459, 259–294 (2009)

  9. 9.

    et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 505, 218–222 (2014)

  10. 10.

    , & Conformational flexibility of metazoan fatty acid synthase enables catalysis. Nature Struct. Mol. Biol . 16, 190–197 (2009)

  11. 11.

    , , , & 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)

  12. 12.

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

  13. 13.

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

  14. 14.

    , , & Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase. J. Mol. Biol. 426, 2229–2245 (2014)

  15. 15.

    et al. Crystal structures of dehydratase domains from the curacin polyketide biosynthetic pathway. Structure 18, 94–105 (2010)

  16. 16.

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

  17. 17.

    , , & Structure and stereospecificity of the dehydratase domain from the terminal module of the rifamycin polyketide synthase. Biochemistry 52, 8916–8928 (2013)

  18. 18.

    et al. Structural basis for cyclopropanation by a unique enoyl-acyl carrier protein reductase. Structure 23, 2213–2223 (2015)

  19. 19.

    , , , & Divergence of multimodular polyketide synthases revealed by a didomain structure. Nature Chem. Biol. 8, 615–621 (2012)

  20. 20.

    , , , & Crystal structure of the human fatty acid synthase enoyl-acyl carrier protein-reductase domain complexed with triclosan reveals allosteric protein-protein interface inhibition. J. Biol. Chem. 289, 33287–33295 (2014)

  21. 21.

    A tylosin ketoreductase reveals how chirality is determined in polyketides. Chem. Biol. 14, 898–908 (2007)

  22. 22.

    et al. Structural and stereochemical analysis of a modular polyketide synthase ketoreductase domain required for the generation of a cis-alkene. Chem. Biol. 20, 772–783 (2013)

  23. 23.

    et al. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site. Nature Chem. Biol. 10, 774–779 (2014)

  24. 24.

    et al. Vinylogous chain branching catalysed by a dedicated polyketide synthase module. Nature 502, 124–128 (2013)

  25. 25.

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

  26. 26.

    et al. Freedom and constraint in engineered noncolinear polyketide assembly lines. Chem. Biol. 22, 229–240 (2015)

  27. 27.

    et al. Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: cluster analysis and assignment of functions. Chem. Biol. 11, 87–97 (2004)

  28. 28.

    & Iteration as programmed event during polyketide assembly; molecular analysis of the aureothin biosynthesis gene cluster. Chem. Biol. 10, 1225–1232 (2003)

  29. 29.

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

  30. 30.

    , , , & Iterative chain elongation by a pikromycin monomodular polyketide synthase. J. Am. Chem. Soc. 125, 4682–4683 (2003)

  31. 31.

    , , & 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)

  32. 32.

    Acta Crystallogr. D 66, 125–132 (2010)

  33. 33.

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

  34. 34.

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

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

    et al. BUSTER version 2.10.2 (Global Phasing, 2011)

  39. 39.

    & Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

  40. 40.

    , , & SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003)

  41. 41.

    , & Bias in cross-validated free R factors: mitigation of the effects of non-crystallographic symmetry. Acta Crystallogr. D 62, 227–238 (2006)

  42. 42.

    et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

  43. 43.

    An automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newslett. Protein Crystallogr . 31, 34–38 (1994)

  44. 44.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  45. 45.

    et al. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem. 326, 234–256 (2004)

  46. 46.

    et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 (2012)

  47. 47.

    , & Structure and flexibility within proteins as identified through small angle X-ray scattering. Gen. Physiol. Biophys. 28, 174–189 (2009)

  48. 48.

    & Accurate flexible fitting of high-resolution protein structures to small-angle x-ray scattering data using a coarse-grained model with implicit hydration shell. Biophys. J. 101, 2981–2991 (2011)

  49. 49.

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

  50. 50.

    , , & The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

  51. 51.

    & The structure of a ketoreductase determines the organization of the β-carbon processing enzymes of modular polyketide synthases. Structure 14, 737–748 (2006)

  52. 52.

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

  53. 53.

    & Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

  54. 54.

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

  55. 55.

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

  56. 56.

    et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014)

  57. 57.

    & Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)

  58. 58.

    , , & Integrating protein structural dynamics and evolutionary analysis with Bio3D. BMC Bioinformatics 15, 399 (2014)

  59. 59.

    A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32, 922–923 (1976)

  60. 60.

    et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009)

  61. 61.

    The PyMOL Molecular Graphics System, version 1.7.0.3 (2010)

  62. 62.

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

  63. 63.

    & Computation of tunnels in protein molecules using Delaunay triangulation. J. WSCG 15, 107–114 (2007)

  64. 64.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

  65. 65.

    et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012)

  66. 66.

    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)

  67. 67.

    , , , & Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase. Chem. Biol. 14, 931–943 (2007)

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Acknowledgements

We acknowledge F. Widdel and J. Zedelius for providing gammaproteobacterium HdN1, P. Leadlay and L. Betancor for providing plasmid pETcoco-2A-L1SL2, and EMBL Heidelberg for providing the pETG-10A vector; J. Missimer and A. Menzel for support in SAXS data acquisition and raw data processing; T. Sharpe for analytical ultracentrifugation, A. Mazur for SAXS refinement, and M. Bertoni for support of the homology-based assignment of the oligomeric state of MAS KS–AT. Data were collected at beamlines PXI, PXIII, and cSAXS of PSI; we acknowledge support from the beamline teams. This work was supported by the Swiss National Science Foundation project grants 125357, 138262, 159696 and R’equip grant 145023. D.A.H. acknowledges a fellowship by the Werner-Siemens Foundation.

Author information

Author notes

    • Dominik A. Herbst
    •  & Roman P. Jakob

    These authors contributed equally to this work.

    • Franziska Zähringer

    Present address: F. Hoffmann-La Roche AG, Grenzacherstrasse 124, 4070 Basel, Switzerland.

Affiliations

  1. Department Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland

    • Dominik A. Herbst
    • , Roman P. Jakob
    • , Franziska Zähringer
    •  & Timm Maier

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Contributions

R.P.J. expressed, purified and crystallized MAS, obtained the crystal structure of the condensing region, collected SAXS data and cloned constructs. F.Z. cloned constructs and purified MAS, GpEryA and MsPks. D.A.H. purified MAS, optimized MAS crystallization, determined the structure of the isolated DH domains and the modifying region, collected SAXS data, analysed the data, performed homology modelling, cloned constructs, and wrote the manuscript. T.M. designed and guided research, analysed data, contributed to crystallographic analysis and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Timm Maier.

Extended data

Supplementary information

Videos

  1. 1.

    Conformational variability and coupling in the MAS modifying region

    All dimeric modifying regions of the MAS crystal structure were aligned to the DH domains and combined into one animation. The ER dimer moves in a screw motion with a lateral translation of up to 8.5Å and a rotation of up to 13.6° on the dimeric DH platform. The ER motion is linked to a rotation of the laterally double-tethered ΨKR/KR domains and couples the conformations of the ΨKR/KR across the MAS modifying region dimer. The maximum observed rotation of the ΨKR/KR domains (40.4°) causes an active site distance shift of 10 Å (euclidean space) relative to the DH domain.

  2. 2.

    Condensing region conformations in crystal structures of PKSs and FASs

    Structures of the homologous condensing regions are aligned and animated from MAS over PDB: 2QO3, 2HG4, 4MZ0, 2VZ9 to 3HHD. The superposition indicates a common hinge for rotational mapping of the AT domain positions located in the linker domain (grey). MAS adopts the most linear arrangement of all AT domains, which differs by a rotation of 43.2° around the common hinge from those observed in the crystal structure of human FAS (PDB: 3HHD).

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