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- PDB-7wij: Cryo-EM structure of prenyltransferase domain of Macrophoma phase... -

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Basic information

Entry
Database: PDB / ID: 7wij
TitleCryo-EM structure of prenyltransferase domain of Macrophoma phaseolina macrophomene synthase
ComponentsGeranylgeranyl diphosphate synthase
KeywordsTRANSFERASE / Macrophoma phaseolina / Macrophomene Synthase / prenyltransferase
Function / homologygeranylgeranyl diphosphate synthase / Polyprenyl synthases signature 1. / isoprenoid biosynthetic process / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Isoprenoid synthase domain superfamily / Geranylgeranyl diphosphate synthase
Function and homology information
Biological speciesMacrophomina phaseolina MS6 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsMori, T. / Adachi, N. / Abe, I.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20am0101071 Japan
CitationJournal: Nature / Year: 2022
Title: Discovery of non-squalene triterpenes.
Authors: Hui Tao / Lukas Lauterbach / Guangkai Bian / Rong Chen / Anwei Hou / Takahiro Mori / Shu Cheng / Ben Hu / Li Lu / Xin Mu / Min Li / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / ...Authors: Hui Tao / Lukas Lauterbach / Guangkai Bian / Rong Chen / Anwei Hou / Takahiro Mori / Shu Cheng / Ben Hu / Li Lu / Xin Mu / Min Li / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Xinghuan Wang / Zixin Deng / Ikuro Abe / Jeroen S Dickschat / Tiangang Liu /
Abstract: All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of short-chain (C-C) terpenes that ...All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of short-chain (C-C) terpenes that are formed from polyisoprenyl diphosphates. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.
History
DepositionJan 3, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Geranylgeranyl diphosphate synthase
B: Geranylgeranyl diphosphate synthase
C: Geranylgeranyl diphosphate synthase
D: Geranylgeranyl diphosphate synthase
E: Geranylgeranyl diphosphate synthase
F: Geranylgeranyl diphosphate synthase


Theoretical massNumber of molelcules
Total (without water)482,6566
Polymers482,6566
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16320 Å2
ΔGint-125 kcal/mol
Surface area70670 Å2

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Components

#1: Protein
Geranylgeranyl diphosphate synthase


Mass: 80442.664 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macrophomina phaseolina MS6 (fungus) / Strain: MS6 / Gene: MPH_02178 / Production host: Escherichia coli (E. coli)
References: UniProt: K2SUY0, geranylgeranyl diphosphate synthase

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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: Macrophoma phaseolina macrophomene synthase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Macrophomina phaseolina
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chrolideNaClSodium chloride1
31 mMDTTC4H10O2S21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse
Specimen supportDetails: This grid was washed with acetone prior to use / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 20 second (blot fource 0)

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1888

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
7PHENIX1.19-4158model fittingmaptomodel
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIX1.19-4158model refinementRealspace refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132926 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 35.95 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002412588
ELECTRON MICROSCOPYf_angle_d0.498116986
ELECTRON MICROSCOPYf_chiral_restr0.03432034
ELECTRON MICROSCOPYf_plane_restr0.00282100
ELECTRON MICROSCOPYf_dihedral_angle_d3.37371692

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