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Title | Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch. |
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Journal, issue, pages | Angew Chem Int Ed Engl, Vol. 63, Issue 13, Page e202315674, Year 2024 |
Publish date | Mar 22, 2024 |
Authors | Dan Ye / Yi-Zhen Shao / Wen-Rui Li / Zhen-Jia Cui / Ting Gong / Jin-Ling Yang / Hai-Qiang Wang / Jun-Gui Dai / Ke-Ping Feng / Ming Ma / Shuang-Gang Ma / Yun-Bao Liu / Ping Zhu / Shi-Shan Yu / |
PubMed Abstract | Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs ...Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons. |
External links | Angew Chem Int Ed Engl / PubMed:38327006 |
Methods | EM (single particle) |
Resolution | 3.1 Å |
Structure data | PDB-7xkw: |
Chemicals | ChemComp-FPS: ChemComp-MG: |
Source |
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Keywords | PLANT PROTEIN / (-)-cyperene synthase / cyclization mechanism / directed evolution / AlphaFold2 |