Context-Dependent Chemoselectivity of Aromatic C-Methyltransferases.

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are generally classified as C-, O-, N-, S-, or halide MTs depending on their methyl acceptor. C-MTs catalyze selective methylation reactions of carbon nucleophiles and play a crucial role in the regulation and diversification of natural products. The control of chemoselectivity by these enzymes is poorly understood, especially with respect to the resonance of a nucleophilic neighboring group that activates the carbon methylation site. We investigated two aromatic C-MTs for the underlying mechanisms governing their chemo- and/or regioselectivity. The unprecedented in vitro dimethylation activity of SfmM2 and NapB5 was demonstrated using the native substrate l-tyrosine and substrates with a 2,4-dihydroxyacetophenone pattern, respectively. Substrate symmetry and the in situ SAM supply with removal of the competitive inhibitor S-adenosyl-l-homocysteine are favorable for dimethylation activity. Through NapB5 catalysis, we obtained C-(di-)methylated acetylphloroglucinol and flavonoid derivatives. We discovered that NapB5 catalyzes both C- and O-methylation of sterically demanding flavonoids. Here, chemoselectivity was modulated by the geometry of substrate binding through substrate selection or site-directed mutagenesis. Precise positioning of the acceptor nucleophile toward SAM is required to achieve regio- and chemoselectivity despite competing C- and O-nucleophilic sites. Thus, chemoselectivity is context-dependent, which opens new horizons for the diversification of natural products.