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- PDB-9b3t: Octameric prenyltransferase domain of linkerless Fusicoccadiene s... -

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Basic information

Entry
Database: PDB / ID: 9b3t
TitleOctameric prenyltransferase domain of linkerless Fusicoccadiene synthase with C2 symmetry without associated cyclase domains
ComponentsFusicoccadiene synthase
KeywordsTRANSFERASE / Enzyme / terpene / engineered construct
Function / homology
Function and homology information


fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / geranylgeranyl diphosphate synthase activity / isoprenoid biosynthetic process / lyase activity / metal ion binding
Similarity search - Function
Terpene synthase family 2, C-terminal metal binding / Polyprenyl synthases signature 1. / Polyprenyl synthases signature 2. / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Fusicoccadiene synthase
Similarity search - Component
Biological speciesPhomopsis amygdali (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsWenger, E.S. / Schultz, K. / Marmorstein, R. / Christianson, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM56838 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Engineering substrate channeling in a bifunctional terpene synthase.
Authors: Eliott S Wenger / Kollin Schultz / Ronen Marmorstein / David W Christianson /
Abstract: Fusicoccadiene synthase from (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate ...Fusicoccadiene synthase from (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69-residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve channeling efficiency. Moreover, GGPP competition experiments with other diterpene cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600-kD "linkerless" construct, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the PT octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.
History
DepositionMar 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Dec 4, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fusicoccadiene synthase
B: Fusicoccadiene synthase
C: Fusicoccadiene synthase
D: Fusicoccadiene synthase
E: Fusicoccadiene synthase
F: Fusicoccadiene synthase
G: Fusicoccadiene synthase
H: Fusicoccadiene synthase


Theoretical massNumber of molelcules
Total (without water)616,5748
Polymers616,5748
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Fusicoccadiene synthase / FS / Fusicoccin A biosynthetic gene clusters protein 1 / PaDC4:GGS


Mass: 77071.812 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phomopsis amygdali (fungus) / Gene: PaFS, orf1 / Production host: Escherichia coli (E. coli)
References: UniProt: A2PZA5, fusicocca-2,10(14)-diene synthase, geranylgeranyl diphosphate synthase
Has protein modificationN

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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: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase
Type: COMPLEX
Details: Linkerless Fusicoccadiene synthase was generated by replacing the native 70-residue linker region of the bifunctional enzyme with the tripeptide PTQ
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.616 MDa / Experimental value: NO
Source (natural)Organism: Diaporthe amygdali (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2200 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
4100 mMTMAOC3H9NO1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 277 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: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.15 sec. / Electron dose: 43 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5558

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 408639
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220663 / Symmetry type: POINT
RefinementCross valid method: NONE

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