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- PDB-8quo: Cryo-EM structure of coproheme decarboxylase from Corynebacterium... -

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Basic information

Entry
Database: PDB / ID: 8quo
TitleCryo-EM structure of coproheme decarboxylase from Corynebacterium diphtheriae in complex with heme b
ComponentsCoproheme decarboxylase
KeywordsOXIDOREDUCTASE / Heme Binding protein / Heme Biosynthesis / Actinobacteria
Function / homology
Function and homology information


oxidoreductase activity, acting on the CH-CH group of donors, oxygen as acceptor / hydrogen peroxide-dependent heme synthase / heme B biosynthetic process / heme binding / metal ion binding
Similarity search - Function
Heme-dependent peroxidase ChdC/CLD / Chlorite dismutase / Dimeric alpha-beta barrel
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Coproheme decarboxylase
Similarity search - Component
Biological speciesCorynebacterium diphtheriae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsPatil, G. / Guo, Y. / Borek, D. / Hofbauer, S.
Funding support Austria, 3items
OrganizationGrant numberCountry
Austrian Science FundP34934 Austria
Austrian Science FundP36967 Austria
Austrian Science FundW1224 Austria
CitationJournal: Protein Sci / Year: 2025
Title: Insights into the flexibility of the domain-linking loop in actinobacterial coproheme decarboxylase through structures and molecular dynamics simulations.
Authors: Gaurav Patil / Diego Javier Alonso de Armiño / Yirui Guo / Paul G Furtmüller / Dominika Borek / Dario A Estrin / Stefan Hofbauer /
Abstract: Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme ...Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme decarboxylase, which oxidatively transforms two propionate groups into vinyl groups yielding heme b. The catalytic reaction cycle of coproheme decarboxylases exhibits four different states: the apo-form, the substrate (coproheme)-bound form, a transient three-propionate intermediate form (monovinyl, monopropionate deuteroheme; MMD), and the product (heme b)-bound form. In this study, we used cryogenic electron microscopy single-particle reconstruction (cryo-EM SPR) to characterize structurally the apo and heme b-bound forms of actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae. The flexible loop that connects the N-terminal and the C-terminal ferredoxin domains of coproheme decarboxylases plays an important role in interactions between the enzyme and porphyrin molecule. To understand the role of this flexible loop, we performed molecular dynamics simulations on the apo and heme b coproheme decarboxylase from Corynebacterium diphtheriae. Our results are discussed in the context of the published structural information on coproheme-bound and MMD-bound coproheme decarboxylase and with respect to the reaction mechanism. Having structural information of all four enzymatically relevant states helps in understanding structural restraints with a functional impact.
History
DepositionOct 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Coproheme decarboxylase
B: Coproheme decarboxylase
C: Coproheme decarboxylase
D: Coproheme decarboxylase
E: Coproheme decarboxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,52710
Polymers136,4455
Non-polymers3,0825
Water23,1131283
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area20910 Å2
ΔGint-163 kcal/mol
Surface area40890 Å2

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Components

#1: Protein
Coproheme decarboxylase


Mass: 27288.961 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Corynebacterium diphtheriae (bacteria) / Gene: chdC / Plasmid: pD441-NH vector / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6NGV6
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1283 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
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: Coproheme decarboxylase from Corynebacterium diphteriae in complex with heme b
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 132.08 kDa/nm / Experimental value: YES
Source (natural)Organism: Corynebacterium diphtheriae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pD441-NH vector
Buffer solutionpH: 7.5 / Details: 100mM Phosphate buffer
Buffer componentName: Sodium chloride / Formula: NaCl
SpecimenConc.: 18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotting time; 5.0-5.5 s. Blotting force; 18 or 19

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 72 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 1305
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 25 eV / Phase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 112

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7MOLREPmodel fitting
9REFMACmodel refinement
10PHENIXmodel refinement
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 870660 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6XUC

6xuc


Accession code: 6XUC / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 81.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002510199
ELECTRON MICROSCOPYf_angle_d0.558313918
ELECTRON MICROSCOPYf_chiral_restr0.04151371
ELECTRON MICROSCOPYf_plane_restr0.00331835
ELECTRON MICROSCOPYf_dihedral_angle_d8.45231413

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