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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.

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

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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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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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