A Versatile Thioesterase Involved in Dimerization during 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 as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, 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 alpha/beta-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an 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 6 pi-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6 pi-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family. A versatile "gene-within-gene" thioesterase (TE) ysfF encodes YsfF and a nested YsfFHBT. The 4-HBT-like enzyme domain in YsfF and YsfFHBT could accomplish the ring-closure reaction for benzyl ring formation. Notably, YsfF and YsfX together are responsible for ACP-tethered dearomatizing dimerization. Furthermore, Asn301, Glu483, and His566 were found to be active-site residues for both activities. image