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Substrate-assisted enzymatic formation of lysinoalanine in duramycin

Nature Chemical Biology volume 14, pages 928–933 (2018)Cite this article

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Abstract

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a β-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal cross-link. Instead, Hya15 of the substrate makes interactions with Lal, suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

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Fig. 1: Duramycin biosynthesis.
Fig. 2: Structural analysis of DurN.
Fig. 3: Proposed substrate-assisted Lal formation catalyzed by DurN.
Fig. 4: MD simulations indicating conformational changes of the Hya15Asp mutant compared to duramycin.
Fig. 5: GC–MS analysis of derivatized lysinoalanine (Lal) and antimicrobial assay of duramycin and its analogs.

Data availability

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 6C0G, 6C0H, and 6C0Y.

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Acknowledgements

The authors thank L. Huo and D.A. Mitchell (UIUC) for providing plasmids, A. Vladimirovich Ulanov (UIUC Metabolomics Center) for assistance with GC–MS analysis, and K. Brister and the staff at LS-CAT at the Advanced Photon Source (Argonne National Laboratory) for assistance with X-ray crystallography data acquisition. This work was supported by the National Institutes of Health (R37 GM 058822 to W.A.v.d.D. and R01 GM079038 to S.K.N.), and D.G.I. MINECO/FEDER (grants CTQ2015-70524-R and RYC-2013-14706 to G.J.-O. and a predoctoral fellowship to C.D.N.).

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Author notes

  1. These authors contributed equally: Linna An, Dillon P. Cogan.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Linna An & Wilfred A. van der Donk

  2. Department of Biochemistry, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Dillon P. Cogan, Satish K. Nair & Wilfred A. van der Donk

  3. Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, La Rioja, Spain

    Claudio D. Navo & Gonzalo Jiménez-Osés

  4. Center for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Satish K. Nair

  5. Howard Hughes Medical Institute, University of Illinois at Urbana–Champaign, Champaign, IL, USA

    Wilfred A. van der Donk

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Contributions

L.A. performed all biochemical assays, designed and analyzed by L.A. and W.A.v.d.D.; D.P.C. and S.K.N. performed and interpreted all structural studies; C.D.N. and G.J.-O. performed the computational analysis. L.A., D.P.C., S.K.N. and W.A.v.d.D. wrote the manuscript. L.A. and D.P.C. contributed equally to this study.

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Correspondence to Satish K. Nair or Wilfred A. van der Donk.

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An, L., Cogan, D.P., Navo, C.D. et al. Substrate-assisted enzymatic formation of lysinoalanine in duramycin. Nat Chem Biol 14, 928–933 (2018). https://doi.org/10.1038/s41589-018-0122-4

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