Context-Dependent Chemoselectivity of Aromatic C-Methyltransferases

Area of Science:

• Enzymology
• Natural Product Biosynthesis
• Organic Chemistry

Background:

• S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are classified by their methyl acceptor (C, O, N, S, halide).
• C-MTs are vital for natural product regulation and diversity, but their chemoselectivity mechanisms remain poorly understood.
• Neighboring group resonance can activate carbon methylation sites, complicating enzyme control.

Purpose of the Study:

• Investigate the mechanisms governing chemo- and regioselectivity in two aromatic C-MTs.
• Explore the role of substrate structure and enzyme engineering in controlling methylation.
• Discover new catalytic activities and substrate specificities of C-MTs.

Main Methods:

• In vitro enzymatic assays using native and modified substrates.
• Demonstration of dimethylation activity for SfmM2 and NapB5.
• Analysis of substrate symmetry, SAM supply, and S-adenosyl-l-homocysteine (SAH) inhibition.
• Site-directed mutagenesis to probe substrate binding and selectivity.

Main Results:

• Unprecedented in vitro dimethylation activity of SfmM2 (on l-tyrosine) and NapB5 (on 2,4-dihydroxyacetophenone derivatives).
• NapB5 catalyzed C- and O-methylation of sterically hindered flavonoids, producing C-(di-)methylated products.
• Favorable conditions for dimethylation include substrate symmetry and optimized SAM/SAH levels.
• Chemoselectivity is modulated by substrate binding geometry, achieved through substrate selection or mutagenesis.

Conclusions:

• Precise nucleophile positioning relative to SAM is essential for achieving regio- and chemoselectivity in C-MTs.
• Enzyme chemoselectivity is context-dependent, influenced by substrate and binding site geometry.
• These findings offer new strategies for the targeted diversification of natural products.