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Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis

Nature Chemical Biology volume 15, pages 795–802 (2019)Cite this article

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Abstract

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free β-ketoacid that subsequently undergoes decarboxylation.

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Fig. 1: Proposed biosynthetic pathways to pactamycin.
Fig. 2: In vivo evidence for the involvement of 3AP-3OP-ACP in pactamycin biosynthesis.
Fig. 3: Loading of 3ABA to the ACP PtmI and formation of β-ketoacyl-PtmI.
Fig. 4: Biochemical characterization of PtmO.
Fig. 5: In vitro reconstitution of the PKS and the glycosyltransferase proteins involved in pactamycin biosynthesis.
Fig. 6: Characterization of the substrate selectivity of PtmK.

Data availability

All data that support the conclusions are included in the published paper and its Supplementary Information, or are available from the authors on request.

References

  1. Mao, Y., Varoglu, M. & Sherman, D. H. Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564. Chem. Biol. 6, 251–263 (1999).

    Article  CAS  Google Scholar 

  2. Kudo, F., Kasama, Y., Hirayama, T. & Eguchi, T. Cloning of the pactamycin biosynthetic gene cluster and characterization of a crucial glycosyltransferase prior to a unique cyclopentane ring formation. J. Antibiot. 60, 492–503 (2007).

    Article  CAS  Google Scholar 

  3. Ito, T. et al. Deciphering pactamycin biosynthesis and engineered production of new pactamycin analogues. Chem. Bio. Chem. 10, 2253–2265 (2009).

    Article  CAS  Google Scholar 

  4. Rinehart, K. L. Jr., Weller, D. D. & Pearce, C. J. Recent biosynthetic studies on antibiotics. J. Nat. Prod. 43, 1–20 (1980).

    Article  CAS  Google Scholar 

  5. Almabruk, K. H. et al. Mutasynthesis of fluorinated pactamycin analogues and their antimalarial activity. Org. Lett. 15, 1678–1681 (2013).

    Article  CAS  Google Scholar 

  6. Hirayama, A., Eguchi, T. & Kudo, F. A single PLP-dependent enzyme PctV catalyzes the transformation of 3-dehydroshikimate into 3-aminobenzoate in the biosynthesis of pactamycin. Chem. Bio. Chem. 14, 1198–1203 (2013).

    Article  CAS  Google Scholar 

  7. Rinehart, K. L. Jr. Biosynthesis and mutasynthesis of aminocyclitol antibiotics. Jpn J. Antibiot. 32, S32–S46 (1979).

    CAS  PubMed  Google Scholar 

  8. Hirayama, A., Miyanaga, A., Kudo, F. & Eguchi, T. Mechanism-based trapping of the quinonoid intermediate by using the K276R mutant of PLP-dependent 3-aminobenzoate synthase PctV in the biosynthesis of pactamycin. Chem. Bio. Chem. 16, 2484–2490 (2015).

    Article  CAS  Google Scholar 

  9. Abugrain, M. E. et al. Interrogating the tailoring steps of pactamycin biosynthesis and accessing new pactamycin analogues. Chem. Bio. Chem. 17, 1585–1588 (2016).

    Article  CAS  Google Scholar 

  10. Abugrain, M. E., Brumsted, C. J., Osborn, A. R., Philmus, B. & Mahmud, T. A highly promiscuous β-ketoacyl-ACP synthase (KAS) III-like protein is involved in pactamycin biosynthesis. ACS Chem. Biol. 12, 362–366 (2017).

    Article  CAS  Google Scholar 

  11. Bibb, M. J., Sherman, D. H., Omura, S. & Hopwood, D. A. Cloning, sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin. Gene 142, 31–39 (1994).

    Article  CAS  Google Scholar 

  12. Ahlert, J. et al. The calicheamicin gene cluster and its iterative type I enediyne PKS. Science 297, 1173–1176 (2002).

    Article  CAS  Google Scholar 

  13. Mao, Y., Varoglu, M. & Sherman, D. H. Genetic localization and molecular characterization of two key genes (mitAB) required for biosynthesis of the antitumor antibiotic mitomycin C. J. Bacteriol. 181, 2199–2208 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lu, W., Roongsawang, N. & Mahmud, T. Biosynthetic studies and genetic engineering of pactamycin analogs with improved selectivity toward malarial parasites. Chem. Biol. 18, 425–431 (2011).

    Article  CAS  Google Scholar 

  15. Upson, R. H., Haugland, R. P., Malekzadeh, M. N. & Haugland, R. P. A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal. Biochem. 243, 41–45 (1996).

    Article  CAS  Google Scholar 

  16. Yang, J. et al. Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis. Org. Biomol. Chem. 9, 438–449 (2011).

    Article  CAS  Google Scholar 

  17. Jia, X. Y. et al. Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis. Chem. Biol. 13, 575–585 (2006).

    Article  CAS  Google Scholar 

  18. Mondal, S., Hsiao, K. & Goueli, S. A. Utility of adenosine monophosphate detection system for monitoring the activities of diverse enzyme reactions. Assay Drug Dev. Technol. 15, 330–341 (2017).

    Article  CAS  Google Scholar 

  19. Asamizu, S., Yang, J., Almabruk, K. H. & Mahmud, T. Pseudoglycosyltransferase catalyzes nonglycosidic C–N coupling in validamycin a biosynthesis. J. Am. Chem. Soc. 133, 12124–12135 (2011).

    Article  CAS  Google Scholar 

  20. Adams, E. S. & Rinehart, K. L. Directed biosynthesis of 5”-fluoropactamycin in Streptomyces pactum. J. Antibiot. 47, 1456–1465 (1994).

    Article  CAS  Google Scholar 

  21. Schmelz, S. & Naismith, J. H. Adenylate-forming enzymes. Curr. Opin. Struct. Biol. 19, 666–671 (2009).

    Article  CAS  Google Scholar 

  22. August, P. R. et al. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol. 5, 69–79 (1998).

    Article  CAS  Google Scholar 

  23. Yu, T. W. et al. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. Proc. Natl Acad. Sci. USA 99, 7968–7973 (2002).

    Article  CAS  Google Scholar 

  24. Admiraal, S. J., Khosla, C. & Walsh, C. T. A Switch for the transfer of substrate between nonribosomal peptide and polyketide modules of the rifamycin synthetase assembly line. J. Am. Chem. Soc. 125, 13664–13665 (2003).

    Article  CAS  Google Scholar 

  25. Zawada, R. J. & Khosla, C. Domain analysis of the molecular recognition features of aromatic polyketide synthase subunits. J. Biol. Chem. 272, 16184–16188 (1997).

    Article  CAS  Google Scholar 

  26. Sherman, D. H., Kim, E. S., Bibb, M. J. & Hopwood, D. A. Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway. J. Bacteriol. 174, 6184–6190 (1992).

    Article  CAS  Google Scholar 

  27. Lu, W., Alanzi, A. R., Abugrain, M. E., Ito, T. & Mahmud, T. Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum. Appl. Microbiol. Biotechnol. 102, 10589–10601 (2018).

    Article  CAS  Google Scholar 

  28. Hirayama, A., Chu, J., Goto, E., Kudo, F. & Eguchi, T. NAD+-dependent dehydrogenase PctP and pyridoxal 5´-phosphate dependent aminotransferase PctC catalyze the first postglycosylation modification of the sugar intermediate in pactamycin biosynthesis. Chem. Bio. Chem. 19, 126–130 (2018).

    Article  CAS  Google Scholar 

  29. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

    Article  CAS  Google Scholar 

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

  31. Green, M. R. & Sambrook, J. in A Laboratory Manual (eds Inglis, J., Boyle, A. & Gann, A.) Ch 3 (Cold Spring Harbor Laboratory Press, 2012).

  32. Ishikawa, J. & Hotta, K. FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol. Lett. 174, 251–253 (1999).

    Article  CAS  Google Scholar 

  33. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  Google Scholar 

  34. He, Y. et al. Two pHZ1358-derivative vectors for efficient gene knockout in Streptomyces. J. Microbiol. Biotechnol. 20, 678–682 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank M. Zabriskie and B. Philmus for critical reading of this manuscript, W. Lu for performing some preliminary work, L. Yang and J. Morre for providing assistance in protein mass spectrometry analysis and A. DeBarber for high-resolution mass spectrometry measurements. This work was supported by grant nos. GM112068 (to T.M.) and AI129957 (to T.M.) from the National Institute of General Medical Sciences and the National Institute of Allergy and Infectious Diseases, respectively. The content is solely the responsibility of the authors and does not represent the official views of the National Institute of General Medical Sciences, the National Institute of Allergy and Infectious Diseases or the National Institutes of Health (NIH).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA

    Auday A. Eida, Mostafa E. Abugrain & Taifo Mahmud

  2. Department of Chemistry, Oregon State University, Corvallis, OR, USA

    Corey J. Brumsted & Taifo Mahmud

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  1. Auday A. Eida
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Contributions

A.A.E. designed and performed the enzymatic assays and analyzed the data; M.E.A. designed and performed the gene inactivation and complementation experiments and analyzed the data; C.J.B. designed and performed the chemical synthesis; T.M. designed the overall project, analyzed the data and wrote the manuscript.

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Correspondence to Taifo Mahmud.

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Supplementary information

Supplementary Tables 1–6 and Supplementary Figures 1–22

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Synthetic Procedures

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Eida, A.A., Abugrain, M.E., Brumsted, C.J. et al. Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis. Nat Chem Biol 15, 795–802 (2019). https://doi.org/10.1038/s41589-019-0314-6

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