Structural Basis for the Friedel-Crafts Alkylation in Cylindrocyclophane Biosynthesis

Abstract

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 to catalyze 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 crystal structure of CylK has not been disclosed and the catalytic mechanism remains obscure. Herein we report the crystal structures of CylK in its apo form and its complexes with the analogues of its substrate and reaction intermediate. Combining the crystal structures, free energy simulations and the mutagenesis experiments, we proposed a concerted double-activation mechanism, which could explain the regioselectivity and stereospecificity. This work provides a foundation for engineering CylK as a biocatalyst to expand its substrate scope and applications in organic synthesis.