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.

Cryptic phosphorylation in nucleoside natural product biosynthesis

An Addendum to this article was published on 11 August 2021

Abstract

Kinases are annotated in many nucleoside biosynthetic gene clusters but generally are considered responsible only for self-resistance. Here, we report an unexpected 2′-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin pathways. This phosphorylation is a unique cryptic modification as it is introduced in the third of seven steps during aminohexuronic acid (AHA) nucleoside biosynthesis, retained throughout the pathway’s duration, and is removed in the last step of the pathway. Bioinformatic analysis of reported nucleoside biosynthetic gene clusters indicates the presence of cryptic phosphorylation in other pathways and the importance of functional characterization of kinases in nucleoside biosynthetic pathways in general. This study also functionally characterized all of the enzymes responsible for AHA biosynthesis and revealed that AHA is constructed via a unique oxidative C–C bond cleavage reaction. The results indicate a divergent biosynthetic mechanism for three classes of antifungal nucleoside natural products.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Nikkomycin and polyoxin pathways.
Fig. 2: Transformation of 5’-OAP into 2’-OAP by PolQ2 and PolJ.
Fig. 3: Transformation of 2′-OAP into AHAP.
Fig. 4: NikS activity assays with AHA or AHAP as the amine substrate.
Fig. 5: LC–MS characterization of metabolites produced by WT and mutant S. cacaoi and S. tendae.
Fig. 6: Nucleoside natural products predicted to be biosynthesized through cryptic phosphorylation.

Data availability

The authors declare that all the data supporting the findings of this study are available within the paper, Extended Data and Supplementary Information. All plasmids, analytical amounts of reported compounds, and raw data are available upon request. Sequences are deposited at National Center for Biotechnology Information under accession nos.: PolQ2, ABX24486; PolJ, ABX24494; PolK, ABX24493; PolD, ABX24500; NikL, CAC80910; NikM, CAC80911; NikK, CAC80909; NikS, CAC11141.

References

  1. Winn, M., Goss, R. J., Kimura, K. & Bugg, T. D. Antimicrobial nucleoside antibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis. Nat. Prod. Rep. 27, 279–304 (2010).

    CAS  Article  PubMed  Google Scholar 

  2. Isono, K. Nucleoside antibiotics: structure, biological-activity, and biosynthesis. J. Antibiot. 41, 1711–1739 (1988).

    CAS  Article  Google Scholar 

  3. Niu, G. & Tan, H. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology. Trends Microbiol. 23, 110–119 (2015).

    CAS  Article  PubMed  Google Scholar 

  4. Chen, W. Q. et al. Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes. J. Ind. Microbiol Biot. 43, 401–417 (2016).

    CAS  Article  Google Scholar 

  5. Shiraishi, T. & Kuzuyama, T. Recent advances in the biosynthesis of nucleoside antibiotics. J. Antibiot. 72, 913–923 (2019).

    CAS  Article  Google Scholar 

  6. Bruntner, C., Lauer, B., Schwarz, W., Mohrle, V. & Bormann, C. Molecular characterization of co-transcribed genes from Streptomyces tendae Tu901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. Mol. Gen. Genet. 262, 102–114 (1999).

    CAS  PubMed  Google Scholar 

  7. Chen, W. et al. Characterization of the polyoxin biosynthetic gene cluster from Streptomyces cacaoi and engineered production of polyoxin H. J. Biol. Chem. 284, 10627–10638 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Nix, D. E., Swezey, R. R., Hector, R. & Galgiani, J. N. Pharmacokinetics of nikkomycin Z after single rising oral doses. Antimicrob. Agents Ch. 53, 2517–2521 (2009).

    CAS  Article  Google Scholar 

  9. Osada, H. Discovery and applications of nucleoside antibiotics beyond polyoxin. J. Antibiot. 72, 855–864 (2019).

    CAS  Article  Google Scholar 

  10. Hector, R. F. Compounds active against cell walls of medically important fungi. Clin. Microbiol. Rev. 6, 1–21 (1993).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Mohrle, V., Roos, U. & Bormann, C. Identification of cellular proteins involved in nikkomycin production in Streptomyces tendae Tu901. Mol. Microbiol. 15, 561–571 (1995).

    CAS  Article  PubMed  Google Scholar 

  12. Ginj, C., Ruegger, H., Amrhein, N. & Macheroux, P. 3′-Enolpyruvyl-UMP, a novel and unexpected metabolite in nikkomycin biosynthesis. ChemBioChem 6, 1974–1976 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Lilla, E. A. & Yokoyama, K. Carbon extension in peptidylnucleoside biosynthesis by radical SAM enzymes. Nat. Chem. Biol. 12, 905–907 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. He, N. et al. Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics. Chem. Sci. 8, 444–451 (2017).

    CAS  Article  PubMed  Google Scholar 

  15. Lauer, B., Sussmuth, R., Kaiser, D., Jung, G. & Bormann, C. A putative enolpyruvyl transferase gene involved in nikkomycin biosynthesis. J. Antibiot. 53, 385–392 (2000).

    CAS  Article  Google Scholar 

  16. Chen, W., Zeng, H. M. & Tan, H. R. Cloning, sequencing, and function of sanF: a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes. Curr. Microbiol. 41, 312–316 (2000).

    CAS  Article  PubMed  Google Scholar 

  17. Xu, J., Liu, G. & Tan, H. sanC- a novel gene involved in nikkomycin biosynthesis in Streptomyces ansochromogenes. Lett. Appl. Microbiol. 36, 234–238 (2003).

    CAS  Article  PubMed  Google Scholar 

  18. Binter, A. et al. Characterization of the PLP-dependent aminotransferase NikK from Streptomyces tendae and its putative role in nikkomycin biosynthesis. FEBS J. 278, 4122–4135 (2011).

    CAS  Article  PubMed  Google Scholar 

  19. Pao, S. S., Paulsen, I. T. & Saier, M. H. Jr Major facilitator superfamily. Microbiol Mol. Biol. Rev. 62, 1–34 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Isono, K., Crain, P. F. & Mccloskey, J. A. Isolation and structure of octosyl acids. Anhydrooctose uronic acid nucleosides. J. Am. Chem. Soc. 97, 943–945 (1975).

    CAS  Article  Google Scholar 

  21. Schuz, T. C., Fiedler, H. P., Zahner, H., Rieck, M. & Konig, W. A. Metabolic products of microorganisms. 263. Nikkomycins Sz, Sx, Soz and Sox, new intermediates associated to the nikkomycin biosynthesis of Streptomyces tendae. J. Antibiot. 45, 199–206 (1992).

    CAS  Article  Google Scholar 

  22. Hong, H., Samborskyy, M., Zhou, Y. & Leadlay, P. F. C-Nucleoside formation in the biosynthesis of the antifungal malayamycin A. Cell Chem. Biol. 26, 493–501 e495 (2019).

    CAS  Article  PubMed  Google Scholar 

  23. Sosio, M. et al. Analysis of the pseudouridimycin biosynthetic pathway provides insights into the formation of C-nucleoside antibiotics. Cell Chem. Biol. 25, 540–549 e544 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Cui, Z. et al. Self-resistance during muraymycin biosynthesis: a complementary nucleotidyltransferase and phosphotransferase with identical modification sites and distinct temporal order. Antimicrob. Agents Chemother. 62, e00193-18 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  25. Kaysser, L. et al. Identification and manipulation of the caprazamycin gene cluster lead to new simplified liponucleoside antibiotics and give insights into the biosynthetic pathway. J. Biol. Chem. 284, 14987–14996 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Funabashi, M. et al. The biosynthesis of liposidomycin-like A-90289 antibiotics featuring a new type of sulfotransferase. ChemBioChem 11, 184–190 (2010).

    CAS  Article  PubMed  Google Scholar 

  27. Chi, X. et al. The muraminomicin biosynthetic gene cluster and enzymatic formation of the 2-deoxyaminoribosyl appendage. MedChemComm 4, 239–243 (2013).

    CAS  Article  PubMed  Google Scholar 

  28. Shiraishi, T., Hiro, N., Igarashi, M., Nishiyama, M. & Kuzuyama, T. Biosynthesis of the antituberculous agent caprazamycin: Identification of caprazol-3’-phosphate, an unprecedented caprazamycin-related metabolite. J. Gen. Appl. Microbiol. 62, 164–166 (2016).

    CAS  Article  PubMed  Google Scholar 

  29. Cui, Z. et al. Pyridoxal-5'-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis. Nat. Chem. Biol. 16, 904–911 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Liu, Y. et al. Discovery and characterization of the tubercidin biosynthetic pathway from Streptomyces tubercidicus NBRC 13090. Micro. Cell Fact. 17, 131 (2018).

    Article  CAS  Google Scholar 

  31. Yang, Z. et al. Functional and kinetic analysis of the phosphotransferase CapP conferring selective self-resistance to capuramycin antibiotics. J. Biol. Chem. 285, 12899–12905 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O'Connor, S. E. & Walsh, C. T. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005).

    CAS  Article  PubMed  Google Scholar 

  33. Cunin, R., Glansdorff, N., Pierard, A. & Stalon, V. Biosynthesis and metabolism of arginine in bacteria. Microbiol. Rev. 50, 314–352 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Li, Y., Llewellyn, N. M., Giri, R., Huang, F. & Spencer, J. B. Biosynthesis of the unique amino acid side chain of butirosin: possible protective-group chemistry in an acyl carrier protein-mediated pathway. Chem. Biol. 12, 665–675 (2005).

    CAS  Article  PubMed  Google Scholar 

  35. Llewellyn, N. M., Li, Y. & Spencer, J. B. Biosynthesis of butirosin: transfer and deprotection of the unique amino acid side chain. Chem. Biol. 14, 379–386 (2007).

    CAS  Article  PubMed  Google Scholar 

  36. Qi, J. et al. Deciphering carbamoylpolyoxamic acid biosynthesis reveals unusual acetylation cycle associated with tandem reduction and sequential hydroxylation. Cell Chem. Biol. 23, 935–944 (2016).

    CAS  Article  PubMed  Google Scholar 

  37. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Nguyen, H. P. & Yokoyama, K. Characterization of acyl carrier protein-dependent glycosyltransferase in mitomycin c biosynthesis. Biochemistry 58, 2804–2808 (2019).

    CAS  Article  PubMed  Google Scholar 

  39. Ogasawara, Y., Nakagawa, Y., Maruyama, C., Hamano, Y. & Dairi, T. In vitro characterization of MitE and MitB: formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins. Bioorg. Med. Chem. Lett. 29, 2076–2078 (2019).

    CAS  PubMed  Article  Google Scholar 

  40. Lauer, B. et al. Molecular characterization of co-transcribed genes from Streptomyces tendae Tu901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin. Mol. Gen. Genet 264, 662–673 (2001).

    CAS  Article  PubMed  Google Scholar 

  41. Li, Y., Zeng, H. & Tan, H. Cloning, function, and expression of sanS: a gene essential for nikkomycin biosynthesis of Streptomyces ansochromogenes. Curr. Microbiol 49, 128–132 (2004).

    CAS  PubMed  Google Scholar 

  42. Gong, R. et al. An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin. Appl. Environ. Microbiol. 84, e00501-18 (2018).

  43. Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).

  44. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical Streptomyces Genetics (John Innes Foundation, 2000).

  45. Thanapipatsiri, A., Claesen, J., Gomez-Escribano, J. P., Bibb, M. & Thamchaipenet, A. A Streptomyces coelicolor host for the heterologous expression of Type III polyketide synthase genes. Micro. Cell Fact. 14, 145 (2015).

    Article  CAS  Google Scholar 

  46. Hover, B. M., Lilla, E. A. & Yokoyama, K. Mechanistic investigation of cPMP synthase in molybdenum cofactor biosynthesis using an uncleavable substrate analogue. Biochemistry 54, 7229–7236 (2015).

    CAS  Article  PubMed  Google Scholar 

  47. Eschenfeldt, W. H., Lucy, S., Millard, C. S., Joachimiak, A. & Mark, I. D. A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol. Biol. 498, 105–115 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Bierman, M. et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43–49 (1992).

    CAS  Article  PubMed  Google Scholar 

  49. Mo, S. et al. Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP. Appl. Environ. Microbiol. 78, 2249–2255 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Paget, M. S., Chamberlin, L., Atrih, A., Foster, S. J. & Buttner, M. J. Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol. 181, 204–211 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. MacNeil, D. J. et al. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68 (1992).

    CAS  Article  PubMed  Google Scholar 

  52. Doumith, M. et al. Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea. Mol. Gen. Genet. 264, 477–485 (2000).

    CAS  Article  PubMed  Google Scholar 

  53. Hong, H. J., Hutchings, M. I., Hill, L. M. & Buttner, M. J. The role of the novel Fem protein VanK in vancomycin resistance in Streptomyces coelicolor. J. Biol. Chem. 280, 13055–13061 (2005).

    CAS  Article  PubMed  Google Scholar 

  54. Schuz, T. C., Fiedler, H. P., Zahner, H., Rieck, M. & Konig, W. A. Metabolic products of microorganisms. 263. Nikkomycins SZ, SX, SoZ and SoX, new intermediates associated to the nikkomycin biosynthesis of Streptomyces tendae. J. Antibiotics 45, 199–206 (1992).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the Duke University Shared Instruments Core Facility (P. Silinski) and the Duke University Lipidomics Core Facility (Z. Guan) for assistance with the LC–HRMS and LC–HRMS/MS analyses, respectively. We thank the Duke University NMR Spectroscopy Core Facility (B. Bobay, R. Venters and D. Mika) for their assistance with the NMR analyses. We thank A. Stelling for assistance with the FTIR analyses. This work was supported by the Duke University School of Medicine, and National Institute of General Medical Sciences R01 GM115729 (to K.Y.). M.M.D. was supported in part by the Duke Medical Scientist Training Program (T32 GM007171).

Author information

Authors and Affiliations

Authors

Contributions

M.M.D., A.T., H.S. and K.Y. designed the experiments. M.M.D. performed the in vitro enzyme characterizations. M.M.D. performed the biosynthetic intermediate purification and characterization experiments. A.T. and H.S. performed the microbiology and gene knockout experiments. M.M.D. and A.T. analyzed the microbial metabolites. M.M.D., A.T. and K.Y. wrote the manuscript.

Corresponding author

Correspondence to Kenichi Yokoyama.

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.

Extended data

Extended Data Fig. 1 Activity of PolD and PolK with 5′-OAP.

Shown are HPLC chromatograms at 260 nm for a full reaction with PolK, a full reaction with PolD, a control without enzyme, and a control without α-KG. No consumption of 5′-OAP was detected. The results were reproducible in at least two independent assays.

Extended Data Fig. 2 Activity of PolQ2 with EP-UMP.

Shown are HPAEC chromatograms at 260 nm for a control without the enzyme (trace i) and the complete reaction (trace ii). No consumption of EP-UMP is detected. The results were reproducible in at least two independent assays.

Extended Data Fig. 3 Activity of PolD, PolK, and NikM with OABP.

Shown are HPAEC chromatograms at 260 nm for a control without the enzyme (trace i), an assay with PolD (ii), an assay with PolK (iii), and an assay with NikM (iv). No consumption of OABP was observed after 24 hours. The results were reproducible in at least two independent assays.

Extended Data Fig. 4 Activity assays of PolJ, NikK, and PolK with 2’-HAP.

a. PolJ phosphatase (10 μM) was incubated with 2′-HAP (100 μM) in 50 mM Tris pH 8.0 supplemented with 10 mM MgCl2 at 25 °C for 18 hrs. b. NikK aminotransferase (30 μM) was incubated with 2′-HAP (200 μM) in 200 mM Tris pH 9.0 supplemented with 1 mM MgCl2 and 10 mM l-Glu at 25 °C for 2 hrs. c. PolK oxygenase (25 μM) was incubated with 2′-HAP (200 μM) in 150 mM NaCl, 50 mM Tris pH 7.5 supplemented with 1 mM Fe2+, 1 mM ascorbate, 200 μM α-KG at 25 °C for 18 hrs. No product formation was detected. The results were reproducible in at least two independent assays.

Extended Data Fig. 5 Activity assays of PolK with KOAP.

PolK (40 μM) was incubated with KOAP (100 μM) in the presence of 100 μM Fe2+, 2 mM ascorbate, and 1 mM α-KG for 20 hours at 25 °C. Even after prolonged incubation (20 hours), no product is observed. The results were reproducible in at least two independent assays.

Extended Data Fig. 6 Activity of PolD with KOAP.

PolD (40 μM) was incubated with KOAP (100 μM) in the presence of 100 μM Fe(II), 1 mM ascorbate, and 2 mM α-KG for 2 hours at 25 °C. No product formation or KOAP consumption. For comparison, under similar conditions, PolD completed the conversion of AHOAP to AHAP in <15 minutes. The results were reproducible in at least two independent assays.

Extended Data Fig. 7 Stereochemistry of AHOAP.

Coupling constants for possible stereochemistry of C4′, C5′, C6′ and C7′. Experimental evidence of JH3′-H4′ = 10.7 Hz, JH4′-H5′ = 3.5 Hz, JH5′-H6′ = 3.5 Hz and JH6′-H7′ = 2.1 Hz for AHOA is most consistent with the stereochemistry of 4′R, 5′R, 6′R, 7′S, indicating that AHOA and AHOAP have 4′R, 5′R, 6′R, 7′S stereochemistry. Dihedral angles were calculated with ChemDraw Professional v19.0 and ChemDraw 3D v19.0 (PerkinElmer Informatics).

Extended Data Fig. 8 Comparison of the rates of reactions between PolD + AHOAP vs. PolD + 2′-OAP.

The PolD assays with AHOAP were performed with 10 μM PolD, 0.1mM Fe2+, 2 mM ascorbate, 100 μM AHOAP in 150 mM NaCl, 50 mM Tris pH 7.6, 10% glycerol. The PolD assays with 2′-OAP were performed with 30μM PolD, 1mM ascorbate, 0.5 mM Fe2+, 200 μM 2’-OAP in 150 mM NaCl, 50 mM Tris pH 7.6.

Extended Data Fig. 9 LC-HRMS analysis of culture media of S. cacaoi ΔpolQ2, wt, and ΔpolQ2 + polQ2.

Shown are EICs (calculated m/z for [M+H]+ ± 5 ppm) for polyoxins A, B, D, F, G, H, I, and J. No polyoxin production was detected in ΔpolQ2 (a), polyoxin A, B, F, and G were found in the wt strain (b), and polyoxin A and F were detected in the ΔpolQ2 + polQ2 strain (c). The observations were reproducible for 2-3 different clones for each mutant strain. The culture was repeated twice for each clone.

Extended Data Fig. 10 Proposed divergent biosynthesis of antifungal nucleoside natural products.

Proposed tailoring of sugar size by oxidative C-C bond cleavage by PolD homologs.

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Figs. 1–16 and Note.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Draelos, M.M., Thanapipatsiri, A., Sucipto, H. et al. Cryptic phosphorylation in nucleoside natural product biosynthesis. Nat Chem Biol 17, 213–221 (2021). https://doi.org/10.1038/s41589-020-00656-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-00656-8

Further reading

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