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Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium

Nature Chemistry volume 9, pages 387–395 (2017)Cite this article

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Abstract

Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning β-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C-methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C-methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature.

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Figure 1: Polytheonamide structures and genetic locus.
Figure 2: Proteolytic results for PTM identification and summary of PoyA mutational analysis.
Figure 3: N-methylation during polytheonamide biosynthesis.
Figure 4: Biosynthetic timing of N-methylation with respect to epimerization.
Figure 5: PoyB- and PoyC-catalysed C-methylation in Rlv3841.
Figure 6: Overview of PTMs observed in polytheonamide biosynthesis.

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Acknowledgements

The authors thank H.-M. Fischer and R. Ledermann for their rhizobia expertise and strains, P. Poole for Rlv3841, M. Frank and L. Arrigo for assistance with PoyI experiments, S. Matsunaga for a T. swinhoei sample, J. LaClair for discussions, and A. Vagstad and H.-M. Fischer for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (31003A_146992 and 205321_165695), the EU (SYNPEPTIDE) and the DFG (PI 430/9-1) (J.P.), the Human Frontier Science Program (M.F.F.), the Studienstiftung des deutschen Volkes (M.J.H.) and the Humboldt Foundation (B.I.M.).

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Authors and Affiliations

  1. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, Zurich, 8093, Switzerland

    Michael F. Freeman, Maximilian J. Helf, Agneya Bhushan, Brandon I. Morinaka & Jörn Piel

  2. Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota–Twin Cities, St Paul, 55108, Minnesota, USA

    Michael F. Freeman

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Contributions

M.F.F., M.H. and A.B. performed all E. coli experiments. M.F.F. designed and performed all rhizobia experiments. M.F.F., M.H., A.B. and B.I.M. performed LC-MS experiments. M.F.F., M.H. and A.B. analysed MS data. J.P., M.F.F. and M.H. designed the studies and wrote the manuscript. All authors discussed the results and made comments on the manuscript.

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Correspondence to Michael F. Freeman or Jörn Piel.

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Freeman, M., Helf, M., Bhushan, A. et al. Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium. Nature Chem 9, 387–395 (2017). https://doi.org/10.1038/nchem.2666

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