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Post-translational formation of strained cyclophanes in bacteria

Nature Chemistry volume 12, pages 1042–1053 (2020)Cite this article

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Abstract

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence–function space.

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Fig. 1: Cyclophane natural products, their sources and associated enzymes.
Fig. 2: Maturase systems, biosynthetic gene clusters and strains of origin.
Fig. 3: Detection and characterization of the Xnc product.
Fig. 4: Detection of activity and characterization of modifications by OscB.
Fig. 5: Protein sequence similarity network for selected SPASM protein families.
Fig. 6: Detection of activity and characterization of the MscB product.
Fig. 7: Production of xenorceptide derived from the xnc gene cluster.

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The data analysed and used to support this study can be found within the main text, supporting information or from the corresponding author upon reasonable request.

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Acknowledgements

We thank J. Wu and Y. Han for assistance with NMR experiments, H. Y. Chen and I. Y. F. Koh for assistance in preparation of the MscA variant, C. Kegler and H. B. Bode for the pCEP plasmid and E. coli S17-1λpir donor strain, X. Cai for helpful discussions, E. Danelius and T. Gonen for attempts to elucidate structures by MicroED and J. Piel for critical reading of the manuscript. We acknowledge funding from the Human Frontier Science Program and Japan Society for Promotion of Science to R.S., the Ministry of Education for research scholarships to M.P. and T.P.D.N. and the MOE NUS Start-up Grant (R-148-000-257-133), MOE Tier 1 Grants (R-148-000-258-114 and R-148-000-271-114) and MOE NUS Early Career Research Award (R-148-000-291-133) to B.I.M.

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Authors and Affiliations

  1. Department of Pharmacy, National University of Singapore, Singapore, Singapore

    Thi Quynh Ngoc Nguyen, Yi Wei Tooh, Ryosuke Sugiyama, Thi Phuong Diep Nguyen, Mugilarasi Purushothaman, Li Chuan Leow, Rubin How Sheng Yong, Irene Agatha & Brandon I. Morinaka

  2. Department of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada

    Karyna Hanif

  3. School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore

    Fernaldo R. Winnerdy & Anh Tuân Phan

  4. Institut Pasteur, Collection des Cyanobactéries, Paris, France

    Muriel Gugger

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Contributions

T.Q.N.N., Y.W.T., R.H.S.Y., M.G. and B.I.M. performed bioinformatics and designed the experiments. T.Q.N.N., Y.W.T., T.P.D.N, M.P., L.C.L., K.H., R.H.S.Y., I.A. and B.I.M. performed coexpression experiments and acquired and analysed LC-MS data. T.Q.N.N., T.P.D.N, F.W., A.T.P. and B.I.M acquired and analysed NMR data. R.S. carried out heterologous expression experiments. T.Q.N.N. carried out promoter exchange experiments. T.Q.N.N and B.I.M carried out degradation and synthesis. T.Q.N.N., Y.W.T. and B.I.M. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Brandon I. Morinaka.

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The authors T.Q.N.N, Y.W.T and B.I.M. are the inventors on International Patent Application No. PCT/SG2020/050303 submitted by the National University of Singapore, which covers the use of radical S-adenosylmethionine enzymes for introducing cyclophanes into polypeptides.

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Supplementary methods, spectral data, Figs. 1–46 and Tables 1–8 and 12.

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Supplementary Tables 9–11.

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Nguyen, T.Q.N., Tooh, Y.W., Sugiyama, R. et al. Post-translational formation of strained cyclophanes in bacteria. Nat. Chem. 12, 1042–1053 (2020). https://doi.org/10.1038/s41557-020-0519-z

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