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- PDB-8c0z: CryoEM structure of a tungsten-containing aldehyde oxidoreductase... -

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Basic information

Entry
Database: PDB / ID: 8c0z
TitleCryoEM structure of a tungsten-containing aldehyde oxidoreductase from Aromatoleum aromaticum
Components
  • Aldehyde:ferredoxin oxidoreductase,tungsten-containing
  • Iron-sulfur cluster-binding protein potential subunit of aldehyde oxidoreductase
  • Similar to ferredoxin:NADH oxidoreductases or NADH oxidases,potential subunit of aldehyde oxidoreductase
KeywordsOXIDOREDUCTASE / Tungsten-containing enzyme / nanowires.
Function / homology
Function and homology information


Oxidoreductases; Acting on the aldehyde or oxo group of donors; With an iron-sulfur protein as acceptor / oxidoreductase activity, acting on the aldehyde or oxo group of donors, iron-sulfur protein as acceptor / iron-sulfur cluster binding / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / electron transfer activity / metal ion binding
Similarity search - Function
Aldehyde ferredoxin oxidoreductase, C-terminal / Aldehyde ferredoxin oxidoreductase, N-terminal / Aldehyde ferredoxin oxidoreductase, domain 2 / Aldehyde ferredoxin oxidoreductase, domain 3 / Aldehyde ferredoxin oxidoreductase-like, C-terminal / Aldehyde ferredoxin oxidoreductase, N-terminal domain superfamily / Aldehyde ferredoxin oxidoreductase, domains 2 & 3 / Aldehyde ferredoxin oxidoreductase, N-terminal domain / Aldehyde ferredoxin oxidoreductase, N-terminal domain / 4Fe-4S dicluster domain ...Aldehyde ferredoxin oxidoreductase, C-terminal / Aldehyde ferredoxin oxidoreductase, N-terminal / Aldehyde ferredoxin oxidoreductase, domain 2 / Aldehyde ferredoxin oxidoreductase, domain 3 / Aldehyde ferredoxin oxidoreductase-like, C-terminal / Aldehyde ferredoxin oxidoreductase, N-terminal domain superfamily / Aldehyde ferredoxin oxidoreductase, domains 2 & 3 / Aldehyde ferredoxin oxidoreductase, N-terminal domain / Aldehyde ferredoxin oxidoreductase, N-terminal domain / 4Fe-4S dicluster domain / 4Fe-4S binding domain / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
BENZOIC ACID / FLAVIN-ADENINE DINUCLEOTIDE / IRON/SULFUR CLUSTER / tungsten cofactor / Similar to ferredoxin:NADH oxidoreductases or NADH oxidases,potential subunit of aldehyde oxidoreductase / Aldehyde:ferredoxin oxidoreductase,tungsten-containing / Iron-sulfur cluster-binding protein potential subunit of aldehyde oxidoreductase
Similarity search - Component
Biological speciesAromatoleum aromaticum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsWiniarska, A. / Ramirez-Amador, F. / Hege, D. / Gemmecker, Y. / Prinz, S. / Hochberg, G. / Heider, J. / Szaleniec, M. / Schuller, J.M.
Funding supportEuropean Union, Poland, Germany, 3items
OrganizationGrant numberCountry
European Union (EU)European Union
Polish National Science Centre Poland
German Research Foundation (DFG) Germany
CitationJournal: Sci Adv / Year: 2023
Title: A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.
Authors: Agnieszka Winiarska / Fidel Ramírez-Amador / Dominik Hege / Yvonne Gemmecker / Simone Prinz / Georg Hochberg / Johann Heider / Maciej Szaleniec / Jan Michael Schuller /
Abstract: Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the ...Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium (AOR) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.
History
DepositionDec 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 14, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation_author.identifier_ORCID
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / 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: Aldehyde:ferredoxin oxidoreductase,tungsten-containing
B: Aldehyde:ferredoxin oxidoreductase,tungsten-containing
C: Iron-sulfur cluster-binding protein potential subunit of aldehyde oxidoreductase
E: Similar to ferredoxin:NADH oxidoreductases or NADH oxidases,potential subunit of aldehyde oxidoreductase
F: Iron-sulfur cluster-binding protein potential subunit of aldehyde oxidoreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,35622
Polymers218,7565
Non-polymers6,60017
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Mass photometry experiments
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18450 Å2
ΔGint-307 kcal/mol
Surface area74010 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules ABCFE

#1: Protein Aldehyde:ferredoxin oxidoreductase,tungsten-containing


Mass: 65954.648 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aromatoleum aromaticum (bacteria) / Gene: ebA5005 / Production host: Aromatoleum evansii (bacteria)
References: UniProt: Q5P143, Oxidoreductases; Acting on the aldehyde or oxo group of donors; With an iron-sulfur protein as acceptor
#2: Protein Iron-sulfur cluster-binding protein potential subunit of aldehyde oxidoreductase


Mass: 20524.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: AorA (chain C,F in the model) is fused to a N-terminal Twin-Strep-tag (two Strep-tags linked by a glycin/serin peptide) for purification purposes.
Source: (gene. exp.) Aromatoleum aromaticum (bacteria) / Gene: ebA5004 / Production host: Aromatoleum evansii (bacteria) / References: UniProt: Q5P144
#3: Protein Similar to ferredoxin:NADH oxidoreductases or NADH oxidases,potential subunit of aldehyde oxidoreductase


Mass: 45798.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aromatoleum aromaticum (bacteria) / Gene: TM0395, ebA5007 / Production host: Aromatoleum evansii (bacteria) / References: UniProt: Q5P142

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Non-polymers , 5 types, 17 molecules

#4: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Fe4S4
#5: Chemical ChemComp-T7R / tungsten cofactor / [(5~{a}~{R},8~{R},9~{a}~{R})-2-azanyl-4-oxidanylidene-8-(phosphonooxymethyl)-6-sulfanyl-3,4~{a},5,5~{a},8,9~{a}-hexahydropyrano[3,2-g]pteridin-7-yl]sulfanyl-[[(5~{a}~{R},8~{R},9~{a}~{R})-2-azanyl-4-oxidanylidene-8-(phosphonooxymethyl)-7-sulfanyl-3,5,5~{a},6,7,8,9~{a},10-octahydropyrano[3,2-g]pteridin-6-yl]sulfanyl]-bis($l^{1}-oxidanyl)tungsten


Mass: 1002.511 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H24N10O14P2S4W / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-BEZ / BENZOIC ACID


Mass: 122.121 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H6O2
#8: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM

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Details

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: Tungsten-containing aldehyde oxidoreductase from Aromatoleum aromaticum
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.304 MDa / Experimental value: YES
Source (natural)Organism: Aromatoleum aromaticum (bacteria)
Source (recombinant)Organism: Aromatoleum evansii (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
145 mMHEPESHEPES1
250 mMsodium chlorideNaCl1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 896

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing-IDNum. of particles selected
11549381
22549381
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
118C0ZC1 (asymmetric)
218C0ZC1 (asymmetric)
328C0ZC1 (asymmetric)
428C0ZC1 (asymmetric)
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
13.22FSC 0.143 CUT-OFF7973118C0ZPOINT
23.22FSC 0.143 CUT-OFF7973118C0ZPOINT
33.22FSC 0.143 CUT-OFF7973128C0ZPOINT
43.22FSC 0.143 CUT-OFF7973128C0ZPOINT
RefinementHighest resolution: 3.22 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315444
ELECTRON MICROSCOPYf_angle_d0.63121049
ELECTRON MICROSCOPYf_dihedral_angle_d5.2462187
ELECTRON MICROSCOPYf_chiral_restr0.0492333
ELECTRON MICROSCOPYf_plane_restr0.0062703

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