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:

Substrate-assisted enzymatic formation of lysinoalanine in duramycin

Nature Chemical Biology volume 14, pages 928–933 (2018)Cite this article

Subjects

Abstract

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a β-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal cross-link. Instead, Hya15 of the substrate makes interactions with Lal, suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Duramycin biosynthesis.
Fig. 2: Structural analysis of DurN.
Fig. 3: Proposed substrate-assisted Lal formation catalyzed by DurN.
Fig. 4: MD simulations indicating conformational changes of the Hya15Asp mutant compared to duramycin.
Fig. 5: GC–MS analysis of derivatized lysinoalanine (Lal) and antimicrobial assay of duramycin and its analogs.

Similar content being viewed by others

Data availability

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 6C0G, 6C0H, and 6C0Y.

References

  1. Kessler, H. et al. The structure of the polycyclic nonadecapeptide Ro 09–0198. Helv. Chim. Acta 71, 1924–1929 (1988).

    Article  CAS  Google Scholar 

  2. Hayashi, F. et al. The structure of PA48009: the revised structure of duramycin. J. Antibiot. (Tokyo) 43, 1421–1430 (1990).

    Article  CAS  Google Scholar 

  3. Fredenhagen, A. et al. Duramycins B and C, two new lanthionine containing antibiotics as inhibitors of phospholipase A2. Structural revision of duramycin and cinnamycin. J. Antibiot. (Tokyo) 43, 1403–1412 (1990).

    Article  CAS  Google Scholar 

  4. Chen, E. et al. Mathermycin, a lantibiotic from the marine actinomycete Marinactinospora thermotolerans SCSIO 00652. Appl. Environ. Microbiol. 83, e00926–17 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Kodani, S., Komaki, H., Ishimura, S., Hemmi, H. & Ohnishi-Kameyama, M. Isolation and structure determination of a new lantibiotic cinnamycin B from Actinomadura atramentaria based on genome mining. J. Ind. Microbiol. Biotechnol. 43, 1159–1165 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Smith, T. E. et al. Accessing chemical diversity from the uncultivated symbionts of small marine animals. Nat. Chem. Biol. 14, 179–185 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pomorski, T., Hrafnsdóttir, S., Devaux, P. F. & van Meer, G. Lipid distribution and transport across cellular membranes. Semin. Cell. Dev. Biol. 12, 139–148 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Iwamoto, K. et al. Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin. Biophys. J. 93, 1608–1619 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hosoda, K. et al. Structure determination of an immunopotentiator peptide, cinnamycin, complexed with lysophosphatidylethanolamine by 1H-NMR1. J. Biochem. 119, 226–230 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Richard, A. S. et al. Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses. Proc. Natl. Acad. Sci. USA 112, 14682–14687 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Oliynyk, I., Varelogianni, G., Roomans, G. M. & Johannesson, M. Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non-cystic fibrosis epithelia. APMIS 118, 982–990 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Zhao, M., Li, Z. & Bugenhagen, S. 99mTc-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe. J. Nucl. Med. 49, 1345–1352 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Delvaeye, T. et al. Noninvasive whole-body imaging of phosphatidylethanolamine as a cell death marker using (99m)Tc-duramycin during TNF-induced SIRS. J. Nucl. Med. 59, 1140–1145 (2018).

    Article  PubMed  Google Scholar 

  14. Huo, L., Ökesli, A., Zhao, M. & van der Donk, W. A. Insights into the biosynthesis of duramycin. Appl. Environ. Microbiol. 83, e02698–16 (2017).

    PubMed  PubMed Central  Google Scholar 

  15. Ökesli, A., Cooper, L. E., Fogle, E. J. & van der Donk, W. A. Nine post-translational modifications during the biosynthesis of cinnamycin. J. Am. Chem. Soc. 133, 13753–13760 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Burley, S. K. et al. RCSB Protein Data Bank: sustaining a living digital data resource that enables breakthroughs in scientific research and biomedical education. Protein Sci. 27, 316–330 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004).

    Article  PubMed  CAS  Google Scholar 

  19. Tang, W., Jiménez-Osés, G., Houk, K. N. & van der Donk, W. A. Substrate control in stereoselective lanthionine biosynthesis. Nat. Chem. 7, 57–64 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Lopatniuk, M., Myronovskyi, M. & Luzhetskyy, A. Streptomyces albus: A new cell factory for non-canonical amino acids incorporation into ribosomally synthesized natural products. ACS. Chem. Biol. 12, 2362–2370 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. O’Rourke, S., Widdick, D. & Bibb, M. A novel mechanism of immunity controls the onset of cinnamycin biosynthesis in Streptomyces cinnamoneus DSM 40646. J. Ind. Microbiol. Biotechnol. 44, 563–572 (2017).

    Article  PubMed  CAS  Google Scholar 

  22. Gerlt, J. A. et al. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta 1854, 1019–1037 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Widdick, D. A. et al. Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005. Proc. Natl. Acad. Sci. USA 100, 4316–4321 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. Lee, S. W. et al. Discovery of a widely distributed toxin biosynthetic gene cluster. Proc. Natl. Acad. Sci. USA 105, 5879–5884 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  28. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65, 1074–1080 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Munson, P. J. & Rodbard, D. An exact correction to the “Cheng-Prusoff” correction. J. Recept. Res. 8, 533–546 (1988).

    Article  CAS  PubMed  Google Scholar 

  34. Götz, A. W. et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized Born. J. Chem. Theory. Comput. 8, 1542–1555 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Pierce, L. C., Salomon-Ferrer, R., Augusto F de Oliveira, C., McCammon, J. A. & Walker, R. C. Routine access to millisecond time scale events with accelerated molecular dynamics. J. Chem. Theory. Comput. 8, 2997–3002 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Le Grand, S., Götz, A. W. & Walker, R. C. SPFP: Speed without compromise-A mixed precision model for GPU accelerated molecular dynamics simulations. Comput. Phys. Commun. 184, 374–380 (2013).

    Article  CAS  Google Scholar 

  37. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Bayly, C. I., Cieplak, P., Cornell, W. D. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  39. Besler, B. H., Merz, K. M. & Kollman, P. A. Atomic charges derived from semiempirical methods. J. Comput. Chem. 11, 431–439 (1990).

    Article  CAS  Google Scholar 

  40. Singh, U. C. & Kollman, P. A. An approach to computing electrostatic charges for molecules. J. Comput. Chem. 5, 129–145 (1984).

    Article  CAS  Google Scholar 

  41. Frisch, M. J. et al. Ver. 16, Revision B.01 (Gaussian, Inc., Wallingford CT, USA, 2016).

  42. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory. Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N-log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank L. Huo and D.A. Mitchell (UIUC) for providing plasmids, A. Vladimirovich Ulanov (UIUC Metabolomics Center) for assistance with GC–MS analysis, and K. Brister and the staff at LS-CAT at the Advanced Photon Source (Argonne National Laboratory) for assistance with X-ray crystallography data acquisition. This work was supported by the National Institutes of Health (R37 GM 058822 to W.A.v.d.D. and R01 GM079038 to S.K.N.), and D.G.I. MINECO/FEDER (grants CTQ2015-70524-R and RYC-2013-14706 to G.J.-O. and a predoctoral fellowship to C.D.N.).

Author information

Author notes

  1. These authors contributed equally: Linna An, Dillon P. Cogan.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Linna An & Wilfred A. van der Donk

  2. Department of Biochemistry, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Dillon P. Cogan, Satish K. Nair & Wilfred A. van der Donk

  3. Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, La Rioja, Spain

    Claudio D. Navo & Gonzalo Jiménez-Osés

  4. Center for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Satish K. Nair

  5. Howard Hughes Medical Institute, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Wilfred A. van der Donk

Authors
  1. Linna An
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dillon P. Cogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Claudio D. Navo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gonzalo Jiménez-Osés
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Satish K. Nair
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wilfred A. van der Donk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.A. performed all biochemical assays, designed and analyzed by L.A. and W.A.v.d.D.; D.P.C. and S.K.N. performed and interpreted all structural studies; C.D.N. and G.J.-O. performed the computational analysis. L.A., D.P.C., S.K.N. and W.A.v.d.D. wrote the manuscript. L.A. and D.P.C. contributed equally to this study.

Corresponding authors

Correspondence to Satish K. Nair or Wilfred A. van der Donk.

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 1–3, Supplementary Figures 1–12

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

An, L., Cogan, D.P., Navo, C.D. et al. Substrate-assisted enzymatic formation of lysinoalanine in duramycin. Nat Chem Biol 14, 928–933 (2018). https://doi.org/10.1038/s41589-018-0122-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0122-4

This article is cited by

Search

Advanced 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