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- PDB-8rwy: DtpB hexamer from Streptomyces lividans -

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Basic information

Entry
Database: PDB / ID: 8rwy
TitleDtpB hexamer from Streptomyces lividans
ComponentsDyp-type peroxidase family
KeywordsOXIDOREDUCTASE / Heme / iron / dye-type peroxidase / METAL BINDING PROTEIN
Function / homology
Function and homology information


peroxidase activity / heme binding / metal ion binding / cytosol
Similarity search - Function
: / : / Dyp-type peroxidase, C-terminal / Dyp-type peroxidase, N-terminal / DyP-type peroxidase family. / Dyp-type peroxidase / Dimeric alpha-beta barrel
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Dyp-type peroxidase family
Similarity search - Component
Biological speciesStreptomyces lividans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsWorrall, J.A.R. / Chaplin, A.K. / Allport, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Protein Sci / Year: 2024
Title: The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.
Authors: Marina Lučić / Thomas Allport / Thomas A Clarke / Lewis J Williams / Michael T Wilson / Amanda K Chaplin / Jonathan A R Worrall /
Abstract: A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet ...A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + β barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.
History
DepositionFeb 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dyp-type peroxidase family
B: Dyp-type peroxidase family
C: Dyp-type peroxidase family
D: Dyp-type peroxidase family
E: Dyp-type peroxidase family
F: Dyp-type peroxidase family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,73212
Polymers205,0336
Non-polymers3,6996
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22120 Å2
ΔGint-230 kcal/mol
Surface area65440 Å2
MethodPISA

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Components

#1: Protein
Dyp-type peroxidase family


Mass: 34172.223 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces lividans (bacteria) / Gene: SSPG_00656 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7U8UU09
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / 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: Hexamer complex of Dye-type peroxidase B from Streptomyces Lividans
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Streptomyces lividans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationCryogen name: ETHANE

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 47.19 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24402 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314450
ELECTRON MICROSCOPYf_angle_d0.47719738
ELECTRON MICROSCOPYf_dihedral_angle_d7.632045
ELECTRON MICROSCOPYf_chiral_restr0.0382169
ELECTRON MICROSCOPYf_plane_restr0.0032608

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