Structural basis for an unprecedented enzymatic alkylation in cylindrocyclophane biosynthesis

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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

1. I-Min A Chen
2. et al
(2021) The IMG/M data management and analysis system v.6.0: new tools and advanced capabilities
IMG-JGI.

https://doi.org/10.1093/nar/gkaa939

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Nathaniel R Braffman

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States

Competing interests
No competing interests declared.
Terry B Ruskoski

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, United States

Competing interests
No competing interests declared.
Katherine M Davis

Department of Chemistry, Pennsylvania State University, University Park, United States

Competing interests
No competing interests declared.
Nathaniel R Glasser

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States

Competing interests
No competing interests declared.
Cassidy Johnson

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States

Competing interests
No competing interests declared.
C Denise Okafor

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, United States

Competing interests
No competing interests declared.
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.
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.

"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.

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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.