Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
Terry B Ruskoski
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States
Katherine M Davis
Department of Chemistry, The Pennsylvania State University, University Park, United States
Nathaniel R Glasser
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
Cassidy Johnson
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
C Denise Okafor
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States; Department of Chemistry, The Pennsylvania State University, University Park, United States
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States; Department of Chemistry, The Pennsylvania State University, University Park, United States
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States; Howard Hughes Medical Institute, Harvard University, Cambridge, United States
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 that these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicate 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.
