Structural Basis for the Friedel–Crafts Alkylation in Cylindrocyclophane Biosynthesis

The Lewis acid-catalyzed Friedel–Crafts alkylation of an aromatic ring with an alkyl halide is extensively used in organic synthesis. However, its biological counterpart was not reported until the elucidation of the cylindrocyclophane biosynthetic pathway in Cylindrospermum licheniforme ATCC 29412 by Balskus and co-workers. CylK is the key enzyme that catalyzes the formation of the cylindrocyclophane scaffold through the Friedel–Crafts alkylation reactions with regioselectivity and stereospecificity. Further research demonstrates that CylK can accept other resorcinol rings and secondary alkyl halides as substrates. To date, the three-dimensional structure of CylK has not been disclosed and the catalytic mechanism remains obscure. Herein we report the crystal structures of CylK alone and complexed with substrate and product analogues. The structures reveal an unprecedented fusion of an RTX-like domain and a seven-bladed β-propeller domain, and the active site architecture that defines the substrate binding mode. Combining the crystal structures, free energy simulations, and the site-directed mutagenesis experiments, we proposed a concerted double-activation mechanism, which could explain the regioselectivity and stereospecificity of this unprecedented enzymatic alkylation consistently. This work provides a foundation for engineering CylK as a biocatalyst to expand its substrate scope and applications in organic synthesis.