Affiliation:
1. State Key Laboratory for Crop Stress Resistance and High-Efficiency Production Shaanxi Key Laboratory of Natural Products & Chemical Biology College of Chemistry & Pharmacy Northwest A&F University Yangling, Shannxi 712100 China
2. Key Laboratory of Marine Drugs Ministry of Education School of Medicine and Pharmacy Ocean University of China Qingdao Shandong 266003 China
3. Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology Qingdao Shandong 266237 China
Abstract
AbstractThe 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 α/β‐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 6π‐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 6π‐electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.
Funder
National Natural Science Foundation of China
Natural Science Foundation of Shandong Province
Fundamental Research Funds for the Central Universities
China Postdoctoral Science Foundation