A Versatile Thioesterase Involved in Dimerizationduring Cinnamoyl Lipid Biosynthesis

The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon‐bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatic dimerization remains unknown. In this work, an unusual“gene‐within‐gene”thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/β‐hydrolase and 4‐hydroxybenzoyl‐CoA thioesterase (4‐HBT)‐like enzyme domains, and a nested YsfFHBT (4‐HBT‐like enzyme). Using intracellular tagged carrier‐protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π‐electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP‐tethered dearomatic dimerization process, possibly via an unprecedent Michael‐type addition reaction. Moreover, site‐directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π‐electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.