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.

β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis

Nature volume 520, pages 383–387 (2015)Cite this article

Subjects

This article has been updated

Abstract

Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks1. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(l-α-aminoadipyl)–l-cysteinyl–d-valine synthetase)2. Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators3,4.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Representative members of the family of β-lactam antibiotics.
Figure 2: Biosynthesis of nocardicin A.
Figure 3: Analysis of the reactions catalysed by module 5.
Figure 4: Proposed β-lactam formation mechanism.

Change history

  • 15 April 2015

    A minor change was made to the main text final paragraph, and a present address added for author N.M.G.

References

  1. Walsh, C. T., O'Brien, R. V. & Khosla, C. Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew. Chem. Int. Ed. 52, 7098–7124 (2013)

    Article  CAS  Google Scholar 

  2. Banko, G., Demain, A. L. & Wolfe, S. δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACV synthetase): a multifunctional enzyme with broad substrate specificity for the synthesis of penicillin and cephalosporin precursors. J. Am. Chem. Soc. 109, 2858–2860 (1987)

    Article  CAS  Google Scholar 

  3. Buller, A. R. & Townsend, C. A. Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad. Proc. Natl Acad. Sci. USA 110, E653–E661 (2013)

    Article  ADS  CAS  Google Scholar 

  4. Galletti, P. & Giacomini, D. Monocyclic β-lactams: new structures for new biological activities. Curr. Med. Chem. 18, 4265–4283 (2011)

    Article  CAS  Google Scholar 

  5. Hamad, B. The antibiotics market. Nature Rev. Drug Discov. 9, 675–676 (2010)

    Article  CAS  Google Scholar 

  6. Demain, A. L. Antibiotics: natural products essential to human health. Med. Res. Rev. 29, 821–842 (2009)

    Article  ADS  CAS  Google Scholar 

  7. McKenna, M. Antibiotic resistance: the last resort. Nature 499, 394–396 (2013)

    Article  ADS  CAS  Google Scholar 

  8. Livermore, D. M. Has the era of untreatable infections arrived? J. Antimicrob. Chemother. 64, i29–i36 (2009)

    Article  CAS  Google Scholar 

  9. Burzlaff, N. I. et al. The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature 401, 721–724 (1999)

    Article  ADS  CAS  Google Scholar 

  10. Bachmann, B. O., Li, R. & Townsend, C. A. β-Lactam synthetase: a new biosynthetic enzyme. Proc. Natl Acad. Sci. USA 95, 9082–9086 (1998)

    Article  ADS  CAS  Google Scholar 

  11. Freeman, M. F., Moshos, K. A., Bodner, M. J., Li, R. & Townsend, C. A. Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis. Proc. Natl Acad. Sci. USA 105, 11128–11133 (2008)

    Article  ADS  CAS  Google Scholar 

  12. Davidsen, J. M. & Townsend, C. A. In vivo characterization of nonribosomal peptide synthetases NocA and NocB in the biosynthesis of nocardicin A. Chem. Biol. 19, 297–306 (2012)

    Article  CAS  Google Scholar 

  13. Davidsen, J. M., Bartley, D. M. & Townsend, C. A. Non-ribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI. J. Am. Chem. Soc. 135, 1749–1759 (2013)

    Article  CAS  Google Scholar 

  14. Reeve, A. M., Breazeale, S. D. & Townsend, C. A. Purification, characterization, and cloning of an S-adenosylmethionine-dependent 3-amino-3-carboxypropyltransferase in nocardicin biosynthesis. J. Biol. Chem. 273, 30695–30703 (1998)

    Article  CAS  Google Scholar 

  15. Townsend, C. A. & Brown, A. M. Nocardicin A: biosynthetic experiments with amino acid precursors. J. Am. Chem. Soc. 105, 913–918 (1983)

    Article  CAS  Google Scholar 

  16. Hubbard, B. K., Thomas, M. G. & Walsh, C. T. Biosynthesis of l- p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics. Chem. Biol. 7, 931–942 (2000)

    Article  CAS  Google Scholar 

  17. Townsend, C. A. & Wilson, B. A. The role of nocardicin G in nocardicin A biosynthesis. J. Am. Chem. Soc. 110, 3320–3321 (1988)

    Article  CAS  Google Scholar 

  18. Gaudelli, N. M. & Townsend, C. A. Epimerization and substrate gating by a TE domain in β-lactam antibiotic biosynthesis. Nature Chem. Biol. 10, 251–258 (2014)

    Article  CAS  Google Scholar 

  19. Walsh, C. T. et al. Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Curr. Opin. Struct. Biol. 5, 525–534 (2001)

    Article  CAS  Google Scholar 

  20. Salituro, G. M. & Townsend, C. A. Total syntheses of (−)-nocardicins A–G: a biogenetic approach. J. Am. Chem. Soc. 112, 760–770 (1990)

    Article  CAS  Google Scholar 

  21. Gunsior, M. et al. The biosynthetic gene cluster for a monocyclic β-lactam antibiotic, nocardicin A. Chem. Biol. 11, 927–938 (2004)

    Article  CAS  Google Scholar 

  22. Belshaw, P. J., Walsh, C. T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999)

    Article  ADS  CAS  Google Scholar 

  23. Quadri, L. E. N. et al. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37, 1585–1595 (1998)

    Article  CAS  Google Scholar 

  24. Roche, E. D. & Walsh, C. T. Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. Biochemistry 42, 1334–1344 (2003)

    Article  CAS  Google Scholar 

  25. Townsend, C. A., Brown, A. M. & Nguyen, L. T. Nocardicin A: stereochemical and biomimetic studies of monocyclic β-lactam formation. J. Am. Chem. Soc. 105, 919–927 (1983)

    Article  CAS  Google Scholar 

  26. Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L.-O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007)

    Article  CAS  Google Scholar 

  27. Tahlan, K. & Jensen, S. E. Origins of the β-lactam rings in natural products. J. Antibiot. (Tokyo) 66, 401–410 (2013)

    Article  CAS  Google Scholar 

  28. Sattely, E. S. & Walsh, C. T. A latent oxazoline electrophile for N-O-C bond formation in pseudomonine biosynthesis. J. Am. Chem. Soc. 130, 12282–12284 (2008)

    Article  CAS  Google Scholar 

  29. Silverman, R. B. The Organic Chemistry of Enzyme-Catalyzed Reactions revised edn, 399–452 (Academic, 2002)

    Google Scholar 

  30. Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013)

    Article  CAS  Google Scholar 

  31. Ehmann, D. E., Trauger, J. W., Stachelhaus, T. & Walsh, C. T. Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases. Chem. Biol. 7, 765–772 (2000)

    Article  CAS  Google Scholar 

  32. Röttig, M. et al. NRPSpredictor2—a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res. 39, W362–W367 (2011)

    Article  Google Scholar 

  33. Rausch, C., Weber, T., Kohlbacher, O., Wohlleben, W. & Huson, D. H. Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res. 33, 5799–5808 (2005)

    Article  CAS  Google Scholar 

  34. Clugston, S. L., Sieber, S. A., Marahiel, M. A. & Walsh, C. T. Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst. Biochemistry 42, 12095–12104 (2003)

    Article  CAS  Google Scholar 

  35. Rausch, C., Hoof, I., Weber, T., Wohlleben, W. & Huson, D. H. Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution. BMC Evol. Biol. 7, 78 (2007)

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institutes of Health grant AI014937. We are indebted to D. W. Udwary (bioinformatics), K. A. Moshos, J. W. Labonte (chemistry) and R.-f. Li (molecular biology) for advice and discussion. We thank C. T. Walsh for the pET29-Sfp expression plasmid, I. P. Mortimer for high-resolution mass spectrometry (HRMS) data and C. Moore for help with NMR acquisitions.

Author information

Author notes

  1. Nicole M. Gaudelli

    Present address: † Present address: Department of Chemistry & Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.,

Authors and Affiliations

  1. Department of Chemistry, Johns Hopkins University, Baltimore, 21218, Maryland, USA

    Nicole M. Gaudelli, Darcie H. Long & Craig A. Townsend

Authors
  1. Nicole M. Gaudelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Darcie H. Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Craig A. Townsend
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.A.T. and N.M.G. developed the hypothesis and designed the study. N.M.G. and D.H.L. performed syntheses and biochemical experiments reported. All authors analysed and discussed the results. N.M.G., D.H.L. and C.A.T. prepared the manuscript.

Corresponding author

Correspondence to Craig A. Townsend.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mass spectrometric verification of apo to holo conversion of PCP4 with Sfp and l-pHPG-l-Arg-d-pHPG-l-Ser-S-coenzyme A (1), forming l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2).

Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic tetrapeptidyl-CoA substrate 1.

Extended Data Figure 2 Mass spectrum of major product generated from reaction catalysed by holo-module 5 supplemented with ATP, l-pHPG and l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2).

The major product observed from the reaction containing l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2), holo-module 5, ATP and l-pHPG was isolated over multiple injections by HPLC (peak isolated indicated in inset HPLC trace) and then characterized by HRMS (ESI+). The exact mass ion corresponding to the [M + H] ion of pro-nocardicin G was observed. Exact mass calculated for pro-nocardicin G: C33H39N8O9: 691.2835; found: 691.2839 [M + H]+.

Extended Data Figure 3 Negative experiments resulting from incubation of holo-module 5 with alternative substrates.

a, Schematic of incubation of the tetrapeptidyl substrate l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and l-pHPG. Pro-nocardicin G was not produced. b, HPLC traces of products obtained after incubation of l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3) and holo-module 5 (+M5(WT)) supplemented with ATP and l-pHPG. Pro-nocardicin G was not observed as verified by comparison with authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3), l-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3), l-pHPG and ATP. (iii) HPLC trace of incubation of l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3), l-pHPG and ATP without enzyme. The peak corresponding to l-pHPG-l-Arg-d-pHPG-l-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c, Schematic of incubation of the dipeptidyl substrate d-pHPG-l-Ser-S-PCP4 (5) with holo-module 5, l-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d, LC–MS traces of products obtained after incubation of d-pHPG-l-Ser-S-PCP4 (5) and holo-module 5 (+M5(WT)) supplemented with ATP and l-pHPG. The corresponding β-lactam product was not observed, as verified by comparison with authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with d-pHPG-l-Ser-S-PCP4 (5), l-pHPG and ATP. (ii) Extracted ion chromatogram of the wild-type reaction in trace (i), the 386.1 m/z ion, corresponding to [M + H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5d-pHPG-l-Ser-S-PCP4 (5), l-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace (iii). (v) Extracted ion chromatogram of authentic standard of nocardicin G.

Extended Data Figure 4 Mass spectrometric comparison of apo with holo conversion of PCP4 with Sfp and d-pHPG-l-Ser-S-coenzyme A (4) forming d-pHPG-l-Ser-S-PCP4 (5).

Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of d-pHPG-l-Ser-S-PCP4 (5) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic dipeptidyl-CoA substrate 4. Owing to diketipiperazine22,31 formation and slow hydrolysis to the unloaded holo-PCP4, the half-life of 5 is approximately 20 min.

Extended Data Figure 5 Multiple sequence alignment of homologous NRPS C domains and the NocB C5 domain.

The alignment was performed using Clustal2 with gap opening and extending penalties of 10 and 0.1 respectively. The alignment included the N-terminal C fragment of EntF, the bacillibactin synthetase DhbF, the tyrocidine synthetase TycB, the surfactin synthetase SrfAC and the naturally free-standing condensation enzyme VibH. Highlighted in a red box is H790 uniquely present in the NocB C5 domain along with the corresponding residues present in the depicted C domains. The NocB C5 domain is N-terminal to the consensus catalytic motif, HHxxxDG, present in all canonical condensation domains.

Extended Data Figure 6 HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2).

a, Schematic of incubation of the tetrapeptidyl substrate l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2) with holo-M5l-pHPG. Pro-nocardicin G was not produced. b, HPLC comparison of unbound products from reaction mixtures containing l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2) and either holo-module 5 or M5l-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2), l-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2), l-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2), l-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.

Extended Data Figure 7 Mass spectral comparison of apo with holo conversion of PCP4 with Sfp and l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-3′-dephospho coenzyme A (6) forming l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7).

Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic eliminated tetrapeptidyl-CoA substrate 6.

Extended Data Figure 8 Mass spectrum of major product generated from reaction catalysed by holo-module 5 and l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7) supplemented with ATP and l-pHPG.

The major product from the reaction containing l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7) was isolated over multiple injections by HPLC (peak indicated in inset HPLC trace) and then determined by HRMS (ESI+). The exact mass ion corresponding to the [M + H]+ ion of pro-nocardicin G was observed. Exact mass calculated for C33H39N8O9: 691.2835; found: 691.2822 [M + H]+.

Extended Data Figure 9 Tandem mass spectrometric comparison of holo-module 5 synthesized pro-nocardicin G from l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2) and l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7).

Fragmentation pattern of holo-module 5 biosynthetic pro-nocardicin G from serine-containing tetrapeptide substrate l-pHPG-l-Arg-d-pHPG-l-Ser-S-PCP4 (2, bottom) is directly comparable to the tandem mass spectrometric fragmentation pattern of holo-module 5 synthesized pro-nocardicin G from the dehydroalanine-containing substrate l-pHPG-l-Arg-d-pHPG-dehydroalanyl- S-PCP4 (7, middle). For comparison, the mass spectrum of synthetic pro-nocardicin G is shown at top.

Extended Data Table 1 Condensation domains with a HHHxxxDG motif and an upstream predicted serine- or threonine-activating A domain
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Synthesis of Substrates, 1H-NMR Spectra of Substrates 1, 3, 4 and 6, Supplementary Tables 1 & 2 and additional references. (PDF 1273 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gaudelli, N., Long, D. & Townsend, C. β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis. Nature 520, 383–387 (2015). https://doi.org/10.1038/nature14100

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature14100

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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