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- PDB-7xkw: The 3D strcuture of (-)-cyperene synthase with substrate analogue FSPP -

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Basic information

Entry
Database: PDB / ID: 7xkw
TitleThe 3D strcuture of (-)-cyperene synthase with substrate analogue FSPP
Components(-)-cyperene synthase
KeywordsPLANT PROTEIN / (-)-cyperene synthase / cyclization mechanism / directed evolution / AlphaFold2
Function / homologyChem-FPS
Function and homology information
Biological speciesArtabotrys hexapetalus (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsYu, S.S. / Zhu, P. / Liu, Y.B. / Ma, S.G. / Ye, D. / Shao, Y.Z. / Li, W.R. / Cui, Z.J.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21732008 China
Other governmentCIFMS-2021-I2M-1-029 China
Other government2018YFA0901900 China
Citation
Journal: Angew Chem Int Ed Engl / Year: 2024
Title: Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.
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 /
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.
#1: Journal: Angew.Chem.Int.Ed.Engl. / Year: 2024
Title: Characterisation and engineering of two highly paralogous sesquiterpene synthases reveal a regioselective reprotonation switch.
Authors: Ye, D. / Shao, Y.Z. / Li, W.R. / Cui, Z.J. / Gong, T. / Yang, J.L. / Wang, H.Q. / Dai, J.G. / Feng, K.P. / Ma, M. / Ma, S.G. / Liu, Y.B. / Zhu, P. / Yu, S.S.
History
DepositionApr 20, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Apr 3, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: (-)-cyperene synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,7135
Polymers65,2411
Non-polymers4714
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein (-)-cyperene synthase


Mass: 65241.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Artabotrys hexapetalus (plant) / Production host: Escherichia coli B (bacteria)
#2: Chemical ChemComp-FPS / S-[(2E,6E)-3,7,11-TRIMETHYLDODECA-2,6,10-TRIENYL] TRIHYDROGEN THIODIPHOSPHATE / FARNESYL THIOPYROPHOSPHATE


Mass: 398.392 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H28O6P2S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of (-)-cyperene synthase with substrate anologue FSPP
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Artabotrys hexapetalus (plant)
Source (recombinant)Organism: Escherichia coli B (bacteria)
Buffer solutionpH: 7.5
Details: 20 uL purified AhCS protein at the concentration of 3 mg/mL was incubated with 0.3uL FSPP (10 mg/mL) on ice for 2h and then the mixture was applied to prepare cryo-EM grids.
Buffer component
IDConc.NameFormulaBuffer-ID
120 2.4trimethylol aminomethane hydrochlorideTris-HCl1
250 2.9sodium chlorideNaCl1
32 0.3dithiothreitolDTT1
42 0.19magnesium chlorideMgCl21
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283635 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024233
ELECTRON MICROSCOPYf_angle_d0.5215720
ELECTRON MICROSCOPYf_dihedral_angle_d5.941582
ELECTRON MICROSCOPYf_chiral_restr0.037632
ELECTRON MICROSCOPYf_plane_restr0.005730

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