S ‐Methyl Thioester Formation Uncovers Early Biosynthetic Steps of Yatakemycin

Sulfur-containing natural products are widely distributed in nature. Yatakemycin (YTM) is a complex antitumor and antifungal antibiotic featuring a typical cyclopropane moiety as the pharmacophore and containing an S-methyl thioester moiety. In this study, functional characterization of three genes (ytkG/F/W) within the biosynthetic gene cluster (BGC) revealed their roles in sulfur transfer and subsequent S-methylation to form the S-methyl thioester moiety. Identification of the sulfur source further suggests that the S-methyl thioester formation is an intersection between primary and secondary metabolic pathways. Additionally, we demonstrate that a key O-methylation step, catalyzed by a catechol-O-methyltransferase (MT)-like MT encoded by a gene outside the BGC, is the prerequisite of the S-methyl thioester formation. Notably, the catalytic mechanism of YtkW, an unprecedented thiocarboxylic acid S-MT, was elucidated via crystal structure determination, molecular docking, and relevant mutagenesis-based analyses. Based on these findings, a plausible early-stage biosynthetic pathway for YTM involving S-methyl thioester formation is proposed, which not only advances the biosynthetic understanding of YTM but also provides promising tools for exploring structural diversification of this antibiotic.