Structural Basis for an Unprecedented Enzymatic Alkylation in Cylindrocyclophane Biosynthesis

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 and Glu374 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.

Structural Basis for the Friedel–Crafts Alkylation in Cylindrocyclophane Biosynthesis

“…The discovery of CylK has not only rationalized the formation of the [7,7]­paracyclophane skeleton in cylindrocyclophane biosynthesis, but also provides a mild, stereospecific, and regioselective method for constructing new aryl–alkyl linkages, which could serve as a powerful tool in organic synthesis and medicinal chemistry. In this work, we report the crystal structures of CylK in its apo form and in complexes with ligands . The crystal structure of CylK is composed of an RTX-like domain and a β-propeller domain, both of which are stabilized by Ca 2+ ions.…”

Biocatalytic Friedel‐Crafts Reactions

“…Two recent studies revealing crystal structures of CylK found it to be a fusion of Ca 2 + binding and β-propeller domains, with multiple Ca 2 + binding sites supporting the fold. [100] Through a combination of structural, mutagenesis and simulation studies, the location of the active site was identified in the dimer interface. Here, critical arginine (R105) and tyrosine (Y473) residues are responsible for enabling chloride abstraction and Figure 7.…”