A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis.

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Youssoufene A1, a dimeric cinnamoyl lipid, shows potent inhibition against multidrug-resistant Enterococcus faecalis.
• The biosynthesis pathway for youssoufene A1, particularly its dearomatization/dimerization, remains unelucidated.

Purpose of the Study:

• To functionally characterize the thioesterase (TE) gene ysfF involved in youssoufene A1 biosynthesis.
• To elucidate the mechanism of dearomatization and dimerization in youssoufene A1 formation.

Main Methods:

• Functional characterization of the "gene-within-gene" thioesterase (TE) gene ysfF.
• Intracellular tagged carrier-protein tracking (ITCT) strategy.
• In vitro reconstitution and in vivo experiments.
• Site-directed mutagenesis.

Main Results:

• The ysfF gene encodes two proteins: full-length YsfF and nested YsfF_HBT, both possessing thioesterase activities.
• YsfF/YsfF_HBT facilitate 6π-electrocyclic ring closure for benzene ring formation.
• YsfF and cyclase YsfX collaborate in ACP-tethered dearomatization/dimerization via a potential Michael-type addition.
• Specific residues (N301, E483, H566) in YsfF are crucial for these reactions.

Conclusions:

• This study reveals the dual role of YsfF in thioesterase activity and facilitating dearomatization/dimerization.
• The findings uncover an unprecedented biosynthetic mechanism involving YsfF and YsfX.
• Enhances understanding of thioesterase (TE) protein family multifunctionality and natural product biosynthesis.