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A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Nature Chemical Biology volume 9, pages 685–692 (2013)Cite this article

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Abstract

Type I polyketide synthases often use programmed β-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where β-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP–HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.

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Figure 1: Biosynthetic pathway for PA-A showing mupirocin H production when HCS is inactivated.
Figure 2: Sequence logos showing sequence conservation in β-branch–associated ACPs and their classification using HMMs.
Figure 3: Solution NMR structures of ACP-mupA3a and ACP-mupA3b.
Figure 4: Helix III packing in type I and II polyketide ACP structures.
Figure 5: Analysis of PA-A production by P. fluorescens NCIMB10586 wild type and ACP substitution strains.
Figure 6: Model for ACP-mupA3a–MupH complex and complementation of P. fluorescens NCIMB10586ΔmupH by MupH homologs.

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References

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

    Article  CAS  PubMed  Google Scholar 

  2. Butler, M.S. Natural products to drugs: natural product–derived compounds in clinical trials. Nat. Prod. Rep. 25, 475–516 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 27, 996–1047 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Calderone, C.T. Isoprenoid-like alkylations in polyketide biosynthesis. Nat. Prod. Rep. 25, 845–853 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Fuller, A.T. et al. Pseudomonic acid: an antibiotic produced by Pseudomonas fluorescens. Nature 234, 416–417 (1971).

    Article  CAS  PubMed  Google Scholar 

  6. Thomas, C.M., Hothersall, J., Willis, C.L. & Simpson, T.J. Resistance to and synthesis of the antibiotic mupirocin. Nat. Rev. Microbiol. 8, 281–289 (2010).

    Article  CAS  PubMed  Google Scholar 

  7. Fukuda, D. et al. A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic. PLoS ONE 6, e18031 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rahman, A.S., Hothersall, J., Crosby, J., Simpson, T.J. & Thomas, C.M. Tandemly duplicated acyl carrier proteins, which increase polyketide antibiotic production, can apparently function either in parallel or in series. J. Biol. Chem. 280, 6399–6408 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Gu, L. et al. Tandem acyl carrier proteins in the curacin biosynthetic pathway promote consecutive multienzyme reactions with a synergistic effect. Angew. Chem. Int. Ed. Engl. 50, 2795–2798 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Busche, A. et al. Characterization of molecular interactions between ACP and halogenase domains in the curacin A polyketide synthase. ACS Chem. Biol. 7, 378–386 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. El-Sayed, A.K. et al. Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586. Chem. Biol. 10, 419–430 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Chang, Z. et al. Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 67, 1356–1367 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Edwards, D.J. et al. Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula. Chem. Biol. 11, 817–833 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Piel, J., Wen, G.P., Platzer, M. & Hui, D.Q. Unprecedented diversity of catalytic domains in the first four modules of the putative pederin polyketide synthase. ChemBioChem 5, 93–98 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Calderone, C.T., Kowtoniuk, W.E., Kelleher, N.L., Walsh, C.T. & Dorrestein, P.C. Convergence of isoprene and polyketide biosynthetic machinery: isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis. Proc. Natl. Acad. Sci. USA 103, 8977–8982 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chen, X.H. et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. J. Bacteriol. 188, 4024–4036 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mattheus, W. et al. Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster. Chem. Biol. 17, 149–159 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Schneider, T.D. & Stephens, R.M. Sequence logos—a new way to display consensus sequences. Nucleic Acids Res. 18, 6097–6100 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Irschik, H. et al. Analysis of the sorangicin gene cluster reinforces the utility of a combined phylogenetic/retrobiosynthetic analysis for deciphering natural product assembly by trans-AT PKS. ChemBioChem 11, 1840–1849 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Nakano, M.M., Corbell, N., Besson, J. & Zuber, P. Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol. Gen. Genet. 232, 313–321 (1992).

    CAS  PubMed  Google Scholar 

  21. Shields, J.A. et al. Phosphopantetheinylation and specificity of acyl carrier proteins in the mupirocin biosynthetic cluster. ChemBioChem 11, 248–255 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Evans, S.E. et al. An ACP structural switch: conformational differences between the apo and holo forms of the actinorhodin polyketide synthase acyl carrier protein. ChemBioChem 9, 2424–2432 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. 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  PubMed  Google Scholar 

  24. Alekseyev, V.Y., Liu, C.W., Cane, D.E., Puglisi, J.D. & Khosla, C. Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase. Protein Sci. 16, 2093–2107 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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  PubMed  Google Scholar 

  26. Crump, M.P. et al. Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2). Biochemistry 36, 6000–6008 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Evans, S.E. et al. Probing the interactions of early polyketide intermediates with the actinorhodin ACP from S. coelicolor A3(2). J. Mol. Biol. 389, 511–528 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. El-Sayed, A.K., Hothersall, J. & Thomas, C.M. Quorum-sensing–dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in Pseudomonas fluorescens NCIMB 10586. Microbiology 147, 2127–2139 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. de Vries, S.J., van Dijk, M. & Bonvin, A.M.J.J. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883–897 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Mihalek, I., Res, I. & Lichtarge, O. A family of evolution-entropy hybrid methods for ranking protein residues by importance. J. Mol. Biol. 336, 1265–1282 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Kufareva, I., Budagyan, L., Raush, E., Totrov, M. & Abagyan, R. PIER: protein interface recognition for structural proteomics. Proteins 67, 400–417 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Valley, C.C. et al. The methionine-aromatic motif plays a unique role in stabilizing protein structure. J. Biol. Chem. 287, 34979–34991 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Calderone, C.T., Iwig, D.F., Dorrestein, P.C., Kelleher, N.L. & Walsh, C.T. Incorporation of nonmethyl branches by isoprenoid-like logic: multiple β-alkylation events in the biosynthesis of myxovirescin A1. Chem. Biol. 14, 835–846 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Khosla, C., Ebertkhosla, S. & Hopwood, D.A. Targeted gene replacements in a Streptomyces polyketide synthase gene-cluster—role for the acyl carrier protein. Mol. Microbiol. 6, 3237–3249 (1992).

    Article  CAS  PubMed  Google Scholar 

  35. Decker, H., Summers, R.G. & Hutchinson, C.R. Overproduction of the acyl carrier protein component of a Type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens. J. Antibiot. (Tokyo) 47, 54–63 (1994).

    Article  CAS  Google Scholar 

  36. Beltran-Alvarez, P., Cox, R.J., Crosby, J. & Simpson, T.J. Dissecting the component reactions catalyzed by the actinorhodin minimal polyketide synthase. Biochemistry 46, 14672–14681 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Daley, D.J. Queuing output processes. Adv. Appl. Prob. 8, 395–415 (1976).

    Article  Google Scholar 

  38. Shiozawa, H. et al. Thiomarinol, a new hybrid antimicrobial antibiotic produced by a marine bacterium fermentation, isolation, structure and antimicrobial activity. J. Antibiot. (Tokyo) 46, 1834–1842 (1993).

    Article  CAS  Google Scholar 

  39. Bertani, G. Studies on lysogenesis. 1. The mode of phage liberation by lysogenic Escherchia coli. J. Bacteriol. 62, 293–300 (1951).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edn. (Cold Spring Harbor Press, New York, 1989).

  41. Lepre, C.A. & Moore, J.M. Microdrop screening: a rapid method to optimize solvent conditions for NMR spectroscopy of proteins. J. Biomol. NMR 12, 493–499 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Vranken, W.F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 (2005).

    CAS  PubMed  Google Scholar 

  43. Ottiger, M., Delaglio, F. & Bax, A. Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J. Magn. Reson. 131, 373–378 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Dosset, P., Hus, J.C., Marion, D. & Blackledge, M. A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings. J. Biomol. NMR 20, 223–231 (2001).

    CAS  PubMed  Google Scholar 

  45. 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  PubMed  Google Scholar 

  46. Habeck, M., Rieping, W., Linge, J.P. & Nilges, M. NOE assignment with ARIA 2.0: the nuts and bolts. Methods Mol. Biol. 278, 379–402 (2004).

    CAS  PubMed  Google Scholar 

  47. Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).

    Article  CAS  PubMed  Google Scholar 

  48. 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 

  49. Spronk, C.A.E.M., Nabuurs, S.B., Krieger, E., Vriend, G. & Vuister, G.W. Validation of protein structures derived by NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 45, 315–337 (2004).

    Article  CAS  Google Scholar 

  50. Bachmann, B.O. & Ravel, J. Methods for in silico prediction of microbial polyketide and non-ribosomal peptide biosynthetic pathways from DNA sequence data in. Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides Vol. 458 (ed. Hopwood, D.A.) 181–217 (Elsevier Inc., 2009).

  51. Letunic, I., Doerks, T. & Bork, P. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 40, D302–D305 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Gu, L. et al. Metabolic coupling of dehydration and decarboxylation in the curacin A pathway: Functional identification of a mechanistically diverse enzyme pair. J. Am. Chem. Soc. 128, 9014–9015 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Eswar, N. et al. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein. Sci. 5, 5.6 (2006).

    Google Scholar 

  54. Joosten, R.P. et al. A series of PDB related databases for everyday needs. Nucleic Acids Res. 39, D411–D419 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Kabsch, W. & Sander, C. Dictionary of protein secondary structure—pattern-recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

    Article  CAS  PubMed  Google Scholar 

  56. Galtier, N., Gouy, M. & Gautier, C. SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 12, 543–548 (1996).

    CAS  PubMed  Google Scholar 

  57. Hornak, V. et al. Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins 65, 712–725 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).

    Article  CAS  PubMed  Google Scholar 

  59. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  PubMed  Google Scholar 

  60. Lindorff-Larsen, K. et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950–1958 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council and the Engineering and Physical Sciences Research Council (BB/F014570/1 to P.W-a. and X.D.; BB/I014373/1 to A.S.H., R.G. and J.H.; EP/F066104/1, BB/I003355/1 and BB/I014039/1 to Z.S. and for LC/MS equipment). E.P. was supported by a European Union studentship grant (FP6-mobility 504501). R.F. and Y.T. were supported by scholarships from the Darwin Trust of Edinburgh. J.M. is supported by a PhD scholarship from the FWO Vlaanderen. P.J.W. and R.F. thank A. Bonvin for technical assistance with HADDOCK.

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  1. Anthony S Haines and Xu Dong: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biosciences, University of Birmingham, Birmingham, UK

    Anthony S Haines, Rohit Farmer, Joanne Hothersall, Elton R Stephens, Erika Yamada, Rachel Gurney, Yuiko Takebayashi, Peter J Winn & Christopher M Thomas

  2. School of Chemistry, Clifton, Bristol, UK

    Xu Dong, Zhongshu Song, Christopher Williams, Eliza Płoskoń, Pakorn Wattana-amorn, Russell J Cox, Christine L Willis, Thomas J Simpson, John Crosby & Matthew P Crump

  3. Division of Gene Technology, KU Leuven, Heverlee, Belgium

    Joleen Masschelein & Rob Lavigne

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Contributions

X.D. purified ACP-mupA3a and ACP-mupA3ab and calculated the NMR structures; P.W.-a. purified ACP mup-A3ab; E.P. purified wild-type ACP mup-A3ab; C.W. calculated NMR structures; C.M.T. and M.P.C. codesigned the study and wrote the paper; J.C. was critical for interpreting data on ACP structure and function; T.J.S. provided expertise in polyketide biosynthesis; A.S.H. discovered the conserved motif, carried out bioinformatic analysis and designed key elements of the experimental tests of our predictions; R.J.C. and C.L.W. designed the LC/MS analysis; Z.S. did the LC/MS analysis; R.F., Y.T. and P.J.W. did bioinformatic analysis and molecular modeling of the ACP mutants and ACP–HCS complex; R.L. and J.M. performed initial BatC complementation assays; and E.Y. and R.G. isolated and characterized the mutant ACP-mupA3a. E.R.S. and J.H. created and characterized the ACP and HCS mutants.

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Correspondence to Christopher M Thomas or Matthew P Crump.

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Haines, A., Dong, X., Song, Z. et al. A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis. Nat Chem Biol 9, 685–692 (2013). https://doi.org/10.1038/nchembio.1342

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