Abstract

The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology and the structure and mechanism of CylK are unknown. Here, we report x-ray crystal structures of CylK, revealing a distinctive fusion of a Ca2+ binding domain and a β-propeller fold. We use a mutagenic screening approach to locate CylK's active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicates Asp440 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.

Data availability

Diffraction data have been deposited in the PDB under the accession codes 7RON, 7ROO. All other data generated or analyzed during this study and included in the manuscript and supporting files; Source Data files have been provided for Figure 4.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Nathaniel R Braffman

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  2. Terry B Ruskoski

    Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, United States
    Competing interests
    No competing interests declared.
  3. Katherine M Davis

    Department of Chemistry, Pennsylvania State University, University Park, United States
    Competing interests
    No competing interests declared.
  4. Nathaniel R Glasser

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  5. Cassidy Johnson

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  6. C Denise Okafor

    Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, United States
    Competing interests
    No competing interests declared.
  7. Amie K Boal

    Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, United States
    For correspondence
    akb20@psu.edu
    Competing interests
    Amie K Boal, Reviewing editor, eLife.
  8. Emily P Balskus

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    For correspondence
    balskus@chemistry.harvard.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5985-5714

Funding

National Science Foundation (1454007)

  • Emily P Balskus

National Science Foundation (2003436)

  • Emily P Balskus

Research Corporation for Science Advancement (Cottrell Scholar Award)

  • Emily P Balskus

National Institutes of Health (GM119707)

  • Amie K Boal

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jungsan Sohn, Johns Hopkins University School of Medicine, United States

Version history

  1. Received: November 22, 2021
  2. Preprint posted: December 2, 2021 (view preprint)
  3. Accepted: February 24, 2022
  4. Accepted Manuscript published: February 25, 2022 (version 1)
  5. Version of Record published: March 11, 2022 (version 2)

Copyright

© 2022, Braffman et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Nathaniel R Braffman
  2. Terry B Ruskoski
  3. Katherine M Davis
  4. Nathaniel R Glasser
  5. Cassidy Johnson
  6. C Denise Okafor
  7. Amie K Boal
  8. Emily P Balskus
(2022)
Structural basis for an unprecedented enzymatic alkylation in cylindrocyclophane biosynthesis
eLife 11:e75761.
https://doi.org/10.7554/eLife.75761

Share this article

https://doi.org/10.7554/eLife.75761

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