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:

An ATP-independent strategy for amide bond formation in antibiotic biosynthesis

Nature Chemical Biology volume 6, pages 581–586 (2010)Cite this article

Subjects

Abstract

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C β-lactamase (CapW) was identified within the biosynthetic gene cluster that—in contrast to the expected β-lactamase activity—catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine–dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond 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

Figure 1: Structure of the capuramycin-related metabolites.
Figure 2: Characterization of CapW as an ester-amide exchange catalyst.
Figure 3: Characterization of CapS as a carboxyl methyltransferase.
Figure 4: Proposed biosynthetic pathway of A-503083s.

Similar content being viewed by others

References

  1. Muramatsu, Y. et al. A-503083 A, B, E and F, novel inhibitors of bacterial translocase I, produced by Streptomyces sp. SANK 62799. J. Antibiot. (Tokyo) 57, 639–646 (2004).

    Article  CAS  Google Scholar 

  2. Muramatsu, Y. et al. Studies on novel bacterial translocase I inhibitors, A-500359s. I. Taxonomy, fermentation, isolation, physic-chemical properties and structure elucidation of A-500359 A, C, D, and G. J. Antibiot. (Tokyo) 56, 243–252 (2003).

    Article  CAS  Google Scholar 

  3. Bugg, T.D.H., Lloyd, A.J. & Roper, D.I. Phospho-MurNAc-pentapeptide translocase (MraY) as a target for antibacterial agents and antibacterial proteins. Infect. Disord. Drug Targets 6, 85–106 (2006).

    Article  CAS  Google Scholar 

  4. Suvorov, M., Fisher, J.F. & Mobashery, S. Bacterial cell wall morphology and biochemistry. in Practical Handbook of Microbiology 2nd edn (eds. Goldman, E. & Green, L.H.) 159–190 (CRC Press, Boca Raton, Florida, USA, 2008).

  5. van Heijenoort, J. Lipid intermediates in the biosynthesis of bacterial peptidoglycan. Microbiol. Mol. Biol. Rev. 71, 620–635 (2007).

    Article  CAS  Google Scholar 

  6. Seto, H. et al. The structure of a new nucleoside antibiotic, capuramycin. Tetrahedr. Lett. 29, 2343–2346 (1988).

    Article  CAS  Google Scholar 

  7. Funabashi, M. et al. Identification of the biosynthetic gene cluster of A-500359s in Streptomyces griseus SANK60196. J. Antibiot. (Tokyo) 62, 325–332 (2009).

    Article  CAS  Google Scholar 

  8. Nolan, E.M. & Walsh, C.T. How nature morphs peptide scaffolds into antibiotics. ChemBioChem 10, 34–53 (2009).

    Article  CAS  Google Scholar 

  9. Fisher, J.F., Meroueh, S.O. & Mobashery, S. Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chem. Rev. 105, 395–424 (2005).

    Article  CAS  Google Scholar 

  10. Lobkovsky, E. et al. Evolution of an enzyme activity: crystallographic structure of 2-Å resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc. Natl. Acad. Sci. USA 90, 11257–11261 (1993).

    Article  CAS  Google Scholar 

  11. Santi, D.V., Webster, R.W. Jr. & Cleland, W.W. Kinetics of aminoacyl-tRNA synthetases catalyzed ATP-PPi exchange. Methods Enzymol. 29, 620–627 (1974).

    Article  CAS  Google Scholar 

  12. Ohnuki, T., Muramatsu, Y., Miyakoshi, S., Takatsu, T. & Inukai, M. Studies on novel bacterial translocase I inhibitors, A-500359s. IV. Biosynthesis of A-500359s. J. Antibiot. (Tokyo) 56, 268–279 (2003).

    Article  CAS  Google Scholar 

  13. Gunstone, F.D. Enzymes as biocatalysts in the modification of natural lipids. J. Sci. Food Agric. 79, 1535–1549 (1999).

    Article  CAS  Google Scholar 

  14. Pratt, R.F. Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins). Cell. Mol. Life Sci. 65, 2138–2155 (2008).

    Article  CAS  Google Scholar 

  15. Prates, J.A.M. et al. The structure of the feruloyl esterase module of xylanase 10B from the Clostridium thermocellum provides insights into substrate recognition. Structure 9, 1183–1190 (2001).

    Article  CAS  Google Scholar 

  16. Matthews, B.W., Sigler, P.B., Henderson, R. & Blow, D.M. Three-dimensional structure of tosyl-α-chymotrypsin. Nature 214, 652–656 (1967).

    Article  CAS  Google Scholar 

  17. Lobkovsky, E. et al. Evolution of an enzyme activity: crystallographic structure of 2-Å resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc. Natl. Acad. Sci. USA 90, 11257–11261 (1993).

    Article  CAS  Google Scholar 

  18. Awakawa, T. et al. Physically discrete β-lactamase-type thioesterase catalyzes product release in atrochrysone synthesis by iterative type I polyketide synthase. Chem. Biol. 16, 613–623 (2009).

    Article  CAS  Google Scholar 

  19. Schneider, K. et al. Macrolactin is the polyketide biosynthesis product of the pks2 cluster of Bacillus amyloliquefaciens FZB42. J. Nat. Prod. 70, 1417–1423 (2007).

    Article  CAS  Google Scholar 

  20. Jensen, S.E. et al. Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 48, 192–202 (2004).

    Article  CAS  Google Scholar 

  21. Fischbach, M.A. & Walsh, C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antiobiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006).

    Article  CAS  Google Scholar 

  22. Koetsier, M.J., Jekel, P.A., van den Berg, M.A., Bovenberg, R.A. & Janssen, D.B. Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum. Biochem. J. 417, 467–476 (2009).

    Article  CAS  Google Scholar 

  23. Tobin, M.B., Fleming, M.D., Skatrud, P.L. & Miller, J.R. Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli. J. Bacteriol. 172, 5908–5914 (1990).

    Article  CAS  Google Scholar 

  24. Schmutz, E. et al. An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489. Eur. J. Biochem. 270, 4413–4419 (2003).

    Article  CAS  Google Scholar 

  25. Kadi, N., Over-Costales, D., Barona-Gomez, F. & Challis, G.L. A new family of ATP-dependent oligomerization-macrocyclization biocatalysts. Nat. Chem. Biol. 3, 652–656 (2007).

    Article  CAS  Google Scholar 

  26. Schmelz, S. et al. AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis. Nat. Chem. Biol. 5, 174–182 (2009).

    Article  CAS  Google Scholar 

  27. Hollenhorst, M.A., Clardy, J. & Walsh, C.T. The ATP-dependent amide ligases DdaG and DdaF assemble the fumaramoyl-dipeptide scaffold of the dapdiamide antibiotics. Biochemistry 48, 10467–10472 (2009).

    Article  CAS  Google Scholar 

  28. Arulanantham, H. et al. ORF17 from the clavulanic acid biosynthesis gene cluster catalyzes the ATP-dependent formation of N-glycyl-clavaminic acid. J. Biol. Chem. 281, 279–287 (2006).

    Article  CAS  Google Scholar 

  29. Gondry, M. et al. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nat. Chem. Biol. 5, 414–420 (2009).

    Article  CAS  Google Scholar 

  30. Griffith, S.C. et al. Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate. J. Mol. Biol. 313, 1103–1116 (2001).

    Article  CAS  Google Scholar 

  31. Cone, M.C., Yin, X., Grochowski, L.L., Parker, M.R. & Zabriskie, T.M. The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes: sequence analysis, organization, and initial characterization. ChemBioChem 4, 821–828 (2003).

    Article  CAS  Google Scholar 

  32. Palaniappan, N., Ayers, S., Gupta, S., Habib, E.-S. & Reynolds, K.A. Production of hygromycin A analogs in Streptomyces hygroscopicus NRRL 2388 through identification and manipulation of the biosynthetic gene cluster. Chem. Biol. 13, 753–764 (2006).

    Article  CAS  Google Scholar 

  33. Saugar, I., Sanz, E., Rubio, M.A., Espinose, J.C. & Jimenez, A. Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus. Eur. J. Biochem. 269, 5527–5535 (2002).

    Article  CAS  Google Scholar 

  34. Sugihara, A. et al. A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of d-amino acid-containing peptides. J. Biochem. 131, 247–254 (2002).

    Article  CAS  Google Scholar 

  35. Muramatsu, Y. et al. Studies on novel bacterial translocase I inhibitors, A-500359s III. Deaminocaprolactam derivatives of capuramycin: A-500359 E, F, H, M-1 and M-2. J. Antibiot. (Tokyo) 56, 259–267 (2003).

    Article  CAS  Google Scholar 

  36. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

  37. Kieser, T., Bibb, M., Buttner, M., Chater, K.F. & Hopwood, D.A. Practical Streptomyces Genetics (The John Innes Foundation, Norwich, UK, 2000).

  38. Van Lanen, S.G. et al. Biosynthesis of the β-amino acid moiety of the enediyne antitumor antibiotic C-1027 featuring β-amino acyl-S-carrier protein intermediates. J. Am. Chem. Soc. 127, 11594–11595 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported in part by the Kentucky Science and Technology Corporation (S.V.L.). We thank G. Elliott and J. Jacobsen (Univ. Kentucky) for technical assistance in mass and NMR spectroscopy.

Author information

Author notes

  1. Masanori Funabashi and Zhaoyong Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Bioengineering Research Group I, Process Technology Research Laboratories, Daiichi Sankyo Co., Ltd., Fukushima, Japan

    Masanori Funabashi, Koichi Nonaka & Masahiko Hosobuchi

  2. Department of Pharmaceutical Sciences, College of Pharmacy, College of Pharmacy, University of Kentucky, Lexington, Kentucky, USA

    Zhaoyong Yang, Xiuling Chi & Steven G Van Lanen

  3. Group V, Exploratory Research Laboratories I, Daiichi Sankyo Co. Ltd., Tokyo, Japan

    Yoko Fujita & Tomoyuki Shibata

Authors
  1. Masanori Funabashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhaoyong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Koichi Nonaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masahiko Hosobuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoko Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tomoyuki Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiuling Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Steven G Van Lanen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.N., M.H., Y.F., T.S. and S.G.V.L. designed the research; M.F., Z.Y., K.N., X.C. and S.G.V.L. performed the experiments; and K.N. and S.G.V.L. wrote the manuscript.

Corresponding authors

Correspondence to Koichi Nonaka or Steven G Van Lanen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–11 (PDF 526 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Funabashi, M., Yang, Z., Nonaka, K. et al. An ATP-independent strategy for amide bond formation in antibiotic biosynthesis. Nat Chem Biol 6, 581–586 (2010). https://doi.org/10.1038/nchembio.393

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.393

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