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

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

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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.

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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.

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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.

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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.

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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

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  2. Darcie H. Long
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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.

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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)

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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

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