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- PDB-8h2t: Cryo-EM structure of IadD/E dioxygenase bound with IAA -

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Basic information

Entry
Database: PDB / ID: 8h2t
TitleCryo-EM structure of IadD/E dioxygenase bound with IAA
Components
  • Aromatic-ring-hydroxylating dioxygenase beta subunit
  • Rieske (2Fe-2S) domain protein
KeywordsFLAVOPROTEIN / heterohexamer / dioxygenase
Function / homology
Function and homology information


3-phenylpropionate catabolic process / dioxygenase activity / 2 iron, 2 sulfur cluster binding / oxidoreductase activity / iron ion binding
Similarity search - Function
Ring hydroxylating beta subunit / Ring-hydroxylating dioxygenase beta subunit / Aromatic-ring-hydroxylating dioxygenase, alpha subunit / Aromatic-ring-hydroxylating dioxygenase, alpha subunit, C-terminal domain / Ring hydroxylating alpha subunit (catalytic domain) / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily / NTF2-like domain superfamily
Similarity search - Domain/homology
: / FE2/S2 (INORGANIC) CLUSTER / 1H-INDOL-3-YLACETIC ACID / Rieske (2Fe-2S) domain protein / Aromatic-ring-hydroxylating dioxygenase beta subunit
Similarity search - Component
Biological speciesVariovorax paradoxus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsYu, G. / Li, Z. / Zhang, H.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071218 China
National Natural Science Foundation of China (NSFC)32200496 China
CitationJournal: PLoS Biol / Year: 2023
Title: Structural and biochemical characterization of the key components of an auxin degradation operon from the rhizosphere bacterium Variovorax.
Authors: Yongjian Ma / Xuzichao Li / Feng Wang / Lingling Zhang / Shengmin Zhou / Xing Che / Dehao Yu / Xiang Liu / Zhuang Li / Huabing Sun / Guimei Yu / Heng Zhang /
Abstract: Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin degradation (iad) operon was recently ...Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin degradation (iad) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax iad operon products and identified 2 proteins, IadK2 and IadD, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that IadK2 is a highly IAA-specific ATP-binding cassette (ABC) transporter solute-binding protein (SBP), likely involved in IAA uptake. IadD interacts with IadE to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene iadC to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of iadC/D/E genes could enable IAA transformation by Escherichia coli, suggesting a promising strategy for repurposing the iad operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.
History
DepositionOct 7, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 3, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Rieske (2Fe-2S) domain protein
A: Aromatic-ring-hydroxylating dioxygenase beta subunit
D: Rieske (2Fe-2S) domain protein
E: Aromatic-ring-hydroxylating dioxygenase beta subunit
G: Rieske (2Fe-2S) domain protein
H: Aromatic-ring-hydroxylating dioxygenase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,04815
Polymers205,8286
Non-polymers1,2219
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Rieske (2Fe-2S) domain protein


Mass: 49656.898 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Variovorax paradoxus (bacteria) / Strain: S110 / Gene: Vapar_1493 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5CSP6
#2: Protein Aromatic-ring-hydroxylating dioxygenase beta subunit


Mass: 18952.352 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Variovorax paradoxus (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5CSP7
#3: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-IAC / 1H-INDOL-3-YLACETIC ACID / INDOLE ACETIC ACID


Mass: 175.184 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H9NO2 / Feature type: SUBJECT OF INVESTIGATION / Comment: hormone*YM
#5: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe / 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: enzyme complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Variovorax paradoxus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: No further treatment.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
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: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2449056
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1000245 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314748
ELECTRON MICROSCOPYf_angle_d0.56119983
ELECTRON MICROSCOPYf_dihedral_angle_d4.6861992
ELECTRON MICROSCOPYf_chiral_restr0.0422079
ELECTRON MICROSCOPYf_plane_restr0.0052634

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