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:

A biosynthetic pathway to aromatic amines that uses glycyl-tRNA as nitrogen donor

Abstract

Aromatic amines in nature are typically installed with Glu or Gln as the nitrogen donor. Here we report a pathway that features glycyl-tRNA instead. During the biosynthesis of pyrroloiminoquinone-type natural products such as ammosamides, peptide-aminoacyl tRNA ligases append amino acids to the C-terminus of a ribosomally synthesized peptide. First, \({\mathrm{Amm}}{{{\mathrm{B}}}}_{{{\mathrm{C}}}}^{{{{\mathrm{Trp}}}}}\) adds Trp in a Trp-tRNA-dependent reaction and the flavoprotein AmmC1 then carries out three hydroxylations of the indole ring of Trp. After oxidation to the corresponding ortho-hydroxy para-quinone, \({\mathrm{Amm}}{{{\mathrm{B}}}}_{{{\mathrm{D}}}}^{{{{\mathrm{Gly}}}}}\) attaches Gly to the indole ring in a Gly-tRNA dependent fashion. Subsequent decarboxylation and hydrolysis results in an amino-substituted indole. Similar transformations are catalysed by orthologous enzymes from Bacillus halodurans. This pathway features three previously unknown biochemical processes using a ribosomally synthesized peptide as scaffold for non-ribosomal peptide extension and chemical modification to generate an amino acid-derived natural product.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Structures of pyrroloiminoquinone-derived natural products and activity of PEARL enzymes.
Fig. 2: Post-translational modifications during ammosamide biosynthesis.
Fig. 3: Conversion of Trp to aminoquinone.
Fig. 4: Known pathways to aromatic amines.

Similar content being viewed by others

Data availability

The raw data associated with the spectra in Figs. 2 and 3, and Supplementary Figs. 35, 7, 8, 1015, 1721 and 2331 were deposited at Mendeley (van der Donk, Wilfred (2021), “PEARL 2021”, Mendeley Data, V1, https://doi.org/10.17632/mk3ttnbt5t.1).

References

  1. Lin, S. et al. Another look at pyrroloiminoquinone alkaloids-perspectives on their therapeutic potential from known structures and semisynthetic analogues. Marine Drugs 15, 98 (2017).

    Article  PubMed Central  Google Scholar 

  2. Peters, S. & Spiteller, P. Sanguinones A and B, blue pyrroloquinoline alkaloids from the fruiting bodies of the mushroom Mycena sanguinolenta. J. Nat. Prod. 70, 1274–1277 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Legentil, L., Benel, L., Bertrand, V., Lesur, B. & Delfourne, E. Synthesis and antitumor characterization of pyrazolic analogues of the marine pyrroloquinoline alkaloids: wakayin and tsitsikammamines. J. Med. Chem. 49, 2979–2988 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Hu, J. F., Fan, H., Xiong, J. & Wu, S. B. Discorhabdins and pyrroloiminoquinone-related alkaloids. Chem. Rev. 111, 5465–5491 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Chen, Q. B., Xin, X. L., Yang, Y., Lee, S. S. & Aisa, H. A. Highly conjugated norditerpenoid and pyrroloquinoline alkaloids with potent PTP1B iinhibitory activity from Nigella glandulifera. J. Nat. Prod. 77, 807–812 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Hughes, C. C., MacMillan, J. B., Gaudencio, S. P., Jensen, P. R. & Fenical, W. The ammosamides: structures of cell cycle modulators from a marine-derived Streptomyces species. Angew. Chem. Int. Ed. 48, 725–727 (2009).

    Article  CAS  Google Scholar 

  7. Jordan, P. A. & Moore, B. S. Biosynthetic pathway connects cryptic ribosomally synthesized posttranslationally modified peptide genes with pyrroloquinoline alkaloids. Cell Chem. Biol. 23, 1504–1514 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Reimer, D. & Hughes, C. C. Thiol-based probe for electrophilic natural products reveals that most of the ammosamides are artifacts. J. Nat. Prod. 80, 126–133 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Reddy, P. V., Banerjee, B. & Cushman, M. Efficient total synthesis of ammosamide B. Org. Lett. 12, 3112–3114 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hughes, C. C., MacMillan, J. B., Gaudencio, S. P., Fenical, W. & La Clair, J. J. Ammosamides A and B target myosin. Angew. Chem. Int. Ed. Engl. 48, 728–732 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Luo, J. et al. Discovery of ammosesters by mining the Streptomyces uncialis DCA2648 genome revealing new insight into ammosamide biosynthesis. J. Ind. Microbiol. Biotechnol. 48, kuab027 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Miyanaga, A. et al. Discovery and assembly-line biosynthesis of the lymphostin pyrroloquinoline alkaloid family of mTOR inhibitors in Salinispora bacteria. J. Am. Chem. Soc. 133, 13311–13313 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Colosimo, D. A. & MacMillan, J. B. Ammosamides unveil novel biosynthetic machinery. Cell Chem. Biol. 23, 1444–1446 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Ting, C. P. et al. Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products. Science 365, 280–284 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ortega, M. A. et al. Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB. Nature 517, 509–512 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Garg, N., Salazar-Ocampo, L. M. & van der Donk, W. A. In vitro activity of the nisin dehydratase NisB. Proc. Natl Acad. Sci. USA 110, 7258–7263 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Repka, L. M., Chekan, J. R., Nair, S. K. & van der Donk, W. A. Mechanistic understanding of lanthipeptide biosynthetic enzymes. Chem. Rev. 117, 5457–5520 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Repka, L. M., Hetrick, K. J., Chee, S. H. & van der Donk, W. A. Characterization of leader peptide binding during catalysis by the nisin dehydratase NisB. J. Am. Chem. Soc. 140, 4200–4203 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bothwell, I. R. et al. Characterization of glutamyl-tRNA-dependent dehydratases using nonreactive substrate mimics. Proc. Natl Acad. Sci. USA 116, 17245–17250 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, Z. & van der Donk, W. A. Nonribosomal peptide extension by a peptide amino-acyl tRNA ligase. J. Am. Chem. Soc. 141, 19625–19633 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hudson, G. A., Zhang, Z., Tietz, J. I., Mitchell, D. A. & van der Donk, W. A. In vitro biosynthesis of the core scaffold of the thiopeptide thiomuracin. J. Am. Chem. Soc. 137, 16012–16015 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bewley, K. D. et al. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation. Proc. Natl Acad. Sci. USA 113, 12450–12455 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sikandar, A., Franz, L., Melse, O., Antes, I. & Koehnke, J. Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis. J. Am. Chem. Soc. 141, 9748–9752 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Severinov, K. & Nair, S. K. Microcin C: biosynthesis and mechanisms of bacterial resistance. Future Microbiol. 7, 281–289 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Allali, N., Afif, H., Couturier, M. & Van Melderen, L. The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation. J. Bacteriol. 184, 3224–3231 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ghilarov, D. et al. The origins of specificity in the microcin-processing protease TldD/E. Structure 25, 1549–1561.e1545 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Burkhart, B. J., Hudson, G. A., Dunbar, K. L. & Mitchell, D. A. A prevalent peptide-binding domain guides ribosomal natural product biosynthesis. Nat. Chem. Biol. 11, 564–570 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ozaki, T. et al. Insights into the biosynthesis of dehydroalanines in goadsporin. ChemBioChem 17, 218–223 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Zhang, Q., Yu, Y., Velásquez, J. E. & van der Donk, W. A. Evolution of lanthipeptide synthetases. Proc. Natl Acad. Sci. USA 109, 18361–18366 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang, M. et al. The non-canonical tetratricopeptide repeat (TPR) domain of fluorescent (FLU) mediates complex formation with glutamyl-tRNA reductase. J. Biol. Chem. 290, 17559–17565 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Perez-Riba, A. & Itzhaki, L. S. The tetratricopeptide-repeat motif is a versatile platform that enables diverse modes of molecular recognition. Curr. Opin. Struct. Biol. 54, 43–49 (2019).

    Article  CAS  PubMed  Google Scholar 

  32. Pallen, M. J., Francis, M. S. & Futterer, K. Tetratricopeptide-like repeats in type-III-secretion chaperones and regulators. FEMS Microbiol. Lett. 223, 53–60 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Das, A. K., Cohen, P. W. & Barford, D. The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein–protein interactions. EMBO J. 17, 1192–1199 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lamb, J. R., Tugendreich, S. & Hieter, P. Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem. Sci 20, 257–259 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Fitzpatrick, P. F. Structural insights into the regulation of aromatic amino acid hydroxylation. Curr. Opin. Struct. Biol. 35, 1–6 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fitzpatrick, P. F. Mechanism of aromatic amino acid hydroxylation. Biochemistry 42, 14083–14091 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Perdivara, I., Deterding, L. J., Przybylski, M. & Tomer, K. B. Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification? J. Am. Soc. Mass. Spectrom. 21, 1114–1117 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Basran, J. et al. The mechanism of formation of N-formylkynurenine by heme dioxygenases. J. Am. Chem. Soc. 133, 16251–16257 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hirose, Y. et al. Involvement of common intermediate 3-hydroxy-l-kynurenine in chromophore biosynthesis of quinomycin family antibiotics. J. Antibiot. 64, 117–122 (2011).

    Article  CAS  Google Scholar 

  40. Todorovski, T., Fedorova, M., Hennig, L. & Hoffmann, R. Synthesis of peptides containing 5-hydroxytryptophan, oxindolylalanine, N-formylkynurenine and kynurenine. J. Pept. Sci. 17, 256–262 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Fuson, R. C. The principle of vinylogy. Chem. Rev. 16, 1–27 (1935).

    Article  CAS  Google Scholar 

  42. Jhulki, I., Chanani, P. K., Abdelwahed, S. H. & Begley, T. P. A remarkable oxidative cascade that replaces the riboflavin C8 methyl with an amino group during roseoflavin biosynthesis. J. Am. Chem. Soc. 138, 8324–8327 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schwarz, J., Konjik, V., Jankowitsch, F., Sandhoff, R. & Mack, M. Identification of the key enzyme of roseoflavin biosynthesis. Angew. Chem. Int. Ed. 55, 6103–6106 (2016).

    Article  CAS  Google Scholar 

  44. Konjik, V. et al. The crystal structure of RosB: insights into the reaction mechanism of the first member of a family of flavodoxin-like enzymes. Angew. Chem. Int. Ed. 56, 1146–1151 (2017).

    Article  CAS  Google Scholar 

  45. Kapoor, I. & Nair, S. K. Structure-guided analyses of a key enzyme involved in the biosynthesis of an antivitamin. Biochemistry 57, 5282–5288 (2018).

    Article  CAS  PubMed  Google Scholar 

  46. Ortega, M. A. et al. Structure and tRNA specificity of MibB, a lantibiotic dehydratase from Actinobacteria involved in NAI-107 biosynthesis. Cell Chem. Biol. 23, 370–380 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Sherlin, L. D. et al. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7, 1671–1678 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Rio, D. C., Ares, M. J., Hannon, G. J. & Nilsen, T. W. RNA: A Laboratory Manual (Cold Spring Harbor Laboratory, 2011).

  49. Walker, S. E. & Fredrick, K. Preparation and evaluation of acylated tRNAs. Methods 44, 81–86 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health (R37 GM058822 to W.v.d.D., T32 GM070421 to P.N.D, F32 GM105297 to R.S., F32 GM129944 to C.P.T., and R01 GM085770 to B.S.M.). We thank M. A. Funk and K.-K. A. Wang for initial attempts to activate the bha cluster and D. A. Berthold for help in purifying GlyQS.

Author information

Authors and Affiliations

Authors

Contributions

P.N.D., H.L., R.A.S., C.P.T., and W.A.v.d.D. designed the study. P.N.D., H.L., R.A.S., C.P.T. and X.Z. performed all experiments. L.Z. acquired and interpreted the NMR data. B.S.M. provided reagents and helpful discussions; P.N.D. and W.A.v.d.D. wrote the manuscript.

Corresponding author

Correspondence to Wilfred A. van der Donk.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemistry thanks Seokhee Kim, A. James Link and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–34, Methods and Tables 1–5.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Daniels, P.N., Lee, H., Splain, R.A. et al. A biosynthetic pathway to aromatic amines that uses glycyl-tRNA as nitrogen donor. Nat. Chem. 14, 71–77 (2022). https://doi.org/10.1038/s41557-021-00802-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-021-00802-2

This article is cited by

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