Abstract
Aromatic polyketide synthase (PKS) products undergo diverse tailoring reactions in the biosynthesis of natural products. S-Adenosyl-l-methionine (SAM)-dependent C-methyltransferases (C-MTs) play a key role in this diversification. In the biosynthesis of napyradiomycins, the C-MT NapB5 from Streptomyces aculeolatus catalyzes the C2 monomethylation of an 1,3,6,8-tetrahydroxynaphthalene (T4HN) building block. Biochemical characterization reveals that NapB5 exhibits chemoselective C-dimethylation activity in vitro, accepting both T4HN and its oxidized derivative flaviolin as substrates. Structure-guided mutagenesis and docking studies suggest that precise substrate positioning governs the enzyme's regio- and chemoselectivity. The proximity between the nucleophilic carbon and the SAM methyl donor is crucial for this selectivity. Furthermore, comparative gene cluster analysis identifies homologous C-MTs in other actinomycetes, underscoring their role in diversifying naphthoquinone-based meroterpenoid natural products.