Biochemicaland Structural Insights of the <i>N</i>‑MethyltransferaseCyaF in Cyanogramide Biosynthesis

<i>N</i>-Methyltransferases involved in indole methylation have seldom been discovered in natural product biosynthesis. This study focuses on the enzyme CyaF, which catalyzes a critical <i>N</i>-methylation step of indole in the β-carboline skeleton during cyanogramide biosynthesis. Seven β-carboline analogues (<b>3</b>–<b>9</b>) were isolated from the recombinant strain <i>Streptomyces coelicolor</i> YF11/<i>cyaABC</i>, including three new compounds (<b>5</b>–<b>7</b>). <i>In vitro</i> assays revealed CyaF’s substrate flexibility. The crystal structure of the CyaF/<i>S</i>-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.