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 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.

Harnessing the potential: advances in cyanobacterial natural product research and biotechnology

“…161 This reaction features sequential, stereo-specic alkylations of resorcinol aromatic rings by the alkyl halide electrophiles. 161,162 Notably, alkylation of aromatic rings with alkyl halides is an important transformation in organic synthesis. However, an enzymatic equivalent was unknown.…”

Accelerating the discovery of alkyl halide-derived natural products using halide depletion

“…As halides are not generally required for bacterial growth 48 , we anticipate that this method can be applied to many other microorganisms that grow in defined low-salt minimal media. In particular, it may prove useful for identifying other products of cryptic halogenation involving CylK homologues, as there are many additional instances of CylK-encoding genes colocalized with CylC-encoding genes in BGCs 49 .…”

Friedel–Crafts reactions for biomolecular chemistry

“…S4, ESI †). 56,57 Plants utilize the formation of the antibiotic 2,4-diacetylphloroglucinol through enzymatic acylation reactions for their disease control against certain bacteria (Scheme 3C). The product of the acylation reaction 2,4-diacetylphloroglucinol (3b) shows antibiotic activity and is of agricultural importance.…”