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- EMDB-34443: cryo-EM structure of a bacterial dioxygenase -

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Basic information

Entry
Database: EMDB / ID: EMD-34443
Titlecryo-EM structure of a bacterial dioxygenase
Map dataCryo-EM structure of a bacterial dioxygenase
Sample
  • Complex: enzyme complex
    • Protein or peptide: Rieske (2Fe-2S) domain protein
    • Protein or peptide: Aromatic-ring-hydroxylating dioxygenase beta subunit
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: 1H-INDOL-3-YLACETIC ACID
  • Ligand: FE (III) ION
Keywordsheterohexamer / dioxygenase / FLAVOPROTEIN
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
Rieske (2Fe-2S) domain protein / Aromatic-ring-hydroxylating dioxygenase beta subunit
Similarity search - Component
Biological speciesVariovorax paradoxus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsYu G / Li Z / Zhang H
Funding support China, 2 items
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, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34443.png

Downloads & links

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Map

FileDownload / File: emd_34443.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of a bacterial dioxygenase
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 4.9
Minimum - Maximum-11.774718 - 21.627376999999999
Average (Standard dev.)-0.000000000003227 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 187.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_34443_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_34443_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : enzyme complex

EntireName: enzyme complex
Components
  • Complex: enzyme complex
    • Protein or peptide: Rieske (2Fe-2S) domain protein
    • Protein or peptide: Aromatic-ring-hydroxylating dioxygenase beta subunit
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: 1H-INDOL-3-YLACETIC ACID
  • Ligand: FE (III) ION

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Supramolecule #1: enzyme complex

SupramoleculeName: enzyme complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Variovorax paradoxus (bacteria)

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

MacromoleculeName: Rieske (2Fe-2S) domain protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Variovorax paradoxus (bacteria) / Strain: S110
Molecular weightTheoretical: 49.656898 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTSYRDNPDA IRALVQDDRV HRDLYTSQEL FELEQEHFFA NTWNYVGHES QLPKPGDWIS NEIAGRPLIV ARHSDGSVRA MMNRCAHKG SRLVNGPCGN TGKFFRCPYH AWTFKTDGSL LAIPLKTGYE NTALHECESA KGLTTLRYVR SHRGFIFVKI S DAGPDFDD ...String:
MTSYRDNPDA IRALVQDDRV HRDLYTSQEL FELEQEHFFA NTWNYVGHES QLPKPGDWIS NEIAGRPLIV ARHSDGSVRA MMNRCAHKG SRLVNGPCGN TGKFFRCPYH AWTFKTDGSL LAIPLKTGYE NTALHECESA KGLTTLRYVR SHRGFIFVKI S DAGPDFDD YFGDSLSSID NMADRSPEGE LEIAGGCLRF MHQCNWKMFV ENLNDTMHPM VAHESSAGTA KRMWADKPED EP KPMAVEQ FAPFMSDYKF FEDMGIRTYD NGHSFTGVHF SIHSKYKAIP AYDDAMKARY GEAKTAQILG MARHNTVYYP NLT IKGAIQ AIRVVKPISA DRTLIESWTF RLKGAPPELL QRTTMYNRLI NSPFSVVGHD DLQAYRGMQA GLHASGNEWV SLHR NYDPS ELKGGEITTG GTNELPMRNQ YRAWVQRMTE TM

UniProtKB: Rieske (2Fe-2S) domain protein

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Macromolecule #2: Aromatic-ring-hydroxylating dioxygenase beta subunit

MacromoleculeName: Aromatic-ring-hydroxylating dioxygenase beta subunit / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Variovorax paradoxus (bacteria)
Molecular weightTheoretical: 18.952352 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MAGTEVTRQD LIDFVVNEAH LLDTRRYEEW NALFTDDAFY WVPLVPDQED GLNHTSHLYE DKLLRELRIE RLKSPRAFSQ QPPSRCHHL LQVPVVEQFD AEGNRFVLRT GFHYTESQGD ELQFYVGTFF HHLTVRDGAL RMTLKRVNLL NCDAALPAVQ L FI

UniProtKB: Aromatic-ring-hydroxylating dioxygenase beta subunit

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Macromolecule #3: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 3 / Number of copies: 3 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #4: 1H-INDOL-3-YLACETIC ACID

MacromoleculeName: 1H-INDOL-3-YLACETIC ACID / type: ligand / ID: 4 / Number of copies: 3 / Formula: IAC
Molecular weightTheoretical: 175.184 Da
Chemical component information

ChemComp-IAC:
1H-INDOL-3-YLACETIC ACID / hormone*YM

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Macromolecule #5: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsNo further treatment.

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Particle selectionNumber selected: 2449056
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1000245
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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