Abstact

N-Methyltransferases involved in indole methylation have seldom been discovered in natural product biosynthesis. This study focuses on the enzyme CyaF, which catalyzes a critical N-methylation step of indole in the beta-carboline skeleton during cyanogramide biosynthesis. Seven beta-carboline analogues (3-9) were isolated from the recombinant strain Streptomyces coelicolor YF11/cyaABC, including three new compounds (5-7). In vitro assays revealed CyaF's substrate flexibility. The crystal structure of the CyaF/S-adenosyl-L-homocysteine (SAH) complex, combined with the AlphaFold-predicted model and site-directed mutagenesis, elucidated the catalytic mechanism and structural features that enable CyaF to accommodate diverse substrates, highlighting its potential for biocatalytic applications.