Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch
Dr. Dan Ye
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorYi-Zhen Shao
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorWen-Rui Li
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorZhen-Jia Cui
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Ting Gong
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Jin-Ling Yang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Hai-Qiang Wang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorProf. Jun-Gui Dai
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Ke-Ping Feng
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorProf. Ming Ma
Department State Key Laboratory of Natural and Biomimetic Drugs, Institution School of Pharmaceutical Sciences, Peking University, Beijing, 100191 People's Republic of China
Search for more papers by this authorCorresponding Author
Dr. Shuang-Gang Ma
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Dr. Yun-Bao Liu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Prof. Ping Zhu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
NHC Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Prof. Shi-Shan Yu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Dan Ye
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorYi-Zhen Shao
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorWen-Rui Li
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
These authors contributed equally.
Search for more papers by this authorZhen-Jia Cui
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Ting Gong
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Jin-Ling Yang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Hai-Qiang Wang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorProf. Jun-Gui Dai
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorDr. Ke-Ping Feng
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorProf. Ming Ma
Department State Key Laboratory of Natural and Biomimetic Drugs, Institution School of Pharmaceutical Sciences, Peking University, Beijing, 100191 People's Republic of China
Search for more papers by this authorCorresponding Author
Dr. Shuang-Gang Ma
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Dr. Yun-Bao Liu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Prof. Ping Zhu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
NHC Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorCorresponding Author
Prof. Shi-Shan Yu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 People's Republic of China
Search for more papers by this authorGraphical Abstract
Three critical active site residues [L311-S419-C458]/[M311-V419-A458] were identified as the switch of the regioselective C3/C6 reprotonation of germacrene A in sesquiterpene synthase catalysis through the characterization and engineering of two highly paralogous sesquiterpene synthases, AhCS and AhIS, using a combination of isotopic labeling experiments, cryo-electron microscopy, and structure-guided mutagenesis.
Abstract
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.
Conflict of interests
The authors declare no conflict of interest.
Open Research
Data Availability Statement
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supporting Information. The gene sequences are deposited in GenBank under the accession numbers ON220888 (AhCS) and ON220889 (AhIS). The cryo-EM maps of the structure of AhCS in complex with FSPP (AhCS-3Mg2+-FSPP) have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code EMD-33209. The atomic models have been deposited in the Protein Data Bank (PDB) with the accession code 7XKW (https://doi.org/10.2210/pdb7XKW/pdb).
Supporting Information
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