S ‐Methyl Thioester Formation Uncovers Early Biosynthetic Steps of Yatakemycin

ABSTRACT 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.