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- PDB-9l1w: 3-phenylpropionate bound dioxygenase HcaE-HcaF -

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Basic information

Entry
Database: PDB / ID: 9l1w
Title3-phenylpropionate bound dioxygenase HcaE-HcaF
Components
  • 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha
  • 3-phenylpropionate/cinnamic acid dioxygenase subunit beta
KeywordsELECTRON TRANSPORT / Rieske-type aromatic dioxygenase
Function / homology
Function and homology information


3-phenylpropanoate dioxygenase / 3-phenylpropionate dioxygenase activity / 3-phenylpropionate dioxygenase complex / 3-phenylpropionate catabolic process / 2 iron, 2 sulfur cluster binding / iron ion binding
Similarity search - Function
3-phenylpropionate/cinnamic acid dioxygenase, alpha subunit / 3-phenylpropionate/cinnamic acid dioxygenase, beta subunit / : / : / Ring-hydroxylating dioxygenase, alpha subunit NdoB-like, C-terminal / 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) ...3-phenylpropionate/cinnamic acid dioxygenase, alpha subunit / 3-phenylpropionate/cinnamic acid dioxygenase, beta subunit / : / : / Ring-hydroxylating dioxygenase, alpha subunit NdoB-like, C-terminal / 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) / Aromatic-ring-hydroxylating dioxygenase, 2Fe-2S-binding site / Bacterial ring hydroxylating dioxygenases alpha-subunit signature. / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulphur 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 / HYDROCINNAMIC ACID / 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha / 3-phenylpropionate/cinnamic acid dioxygenase subunit beta
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsJiang, W.X. / Wu, M. / Cheng, X.Q. / Ma, L.X. / Xing, Q.
Funding support China, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32371277 China
National Science Foundation (NSF, China)32301028 China
CitationJournal: J Hazard Mater / Year: 2025
Title: Structure-guided engineering of a Rieske-type aromatic dioxygenase for enhanced consumption of 3-phenylpropionic acid in Escherichia coli.
Authors: Wenxue Jiang / Miao Wu / Zhou Gong / Linhua Han / Xiaoqi Cheng / Xiaoqin Tang / Xiaolong Yu / Xu Dong / Yibin Cheng / Lixin Ma / Qiong Xing /
Abstract: Industrial derived aromatic hydrocarbons are persistent environmental pollutants due to their chemical stability, posing both ecological and health risks. Rieske-type aromatic dioxygenases (RDOs), ...Industrial derived aromatic hydrocarbons are persistent environmental pollutants due to their chemical stability, posing both ecological and health risks. Rieske-type aromatic dioxygenases (RDOs), known for their role in dihydroxylation of aromatic rings, play a pivotal role in microbial consumption and degradation of such compounds. While the industrial application of these enzymes has been impeded by their instability and low biodegradation rate. In this study, we focused on optimization and application of the Rieske-type dioxygenase HcaEF from Escherichia coli (E. coli) K-12, which initializes the degradation of 3-phenylpropionic acid (3-PP) and cinnamic acid (CI). Using cryo-electron microscopy (cryo-EM), we determined the high-resolution structures of the apo-form and 3-PP bound form of HcaEF, revealing key insights into substrate specificity and thermal stability. Leveraging these structural insights, we engineered a Q73I variant of HcaEF. Upon introduction of this mutation, the turnover rate increased from 29.6 % to 43.8 %, showing ∼50 % improvement. Overexpression of this variant in E. coli K-12 significantly enhanced the strain's ability to utilize 3-PP, demonstrating the potential for microbial engineering in environmental bioremediation and industrial applications. Our findings not only deepen the understanding of substrate recognition in RDOs, but also pave the way for developing high-efficiency enzymes for aromatic compound bio-utilization.
History
DepositionDec 16, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha
B: 3-phenylpropionate/cinnamic acid dioxygenase subunit beta
C: 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha
D: 3-phenylpropionate/cinnamic acid dioxygenase subunit beta
E: 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha
F: 3-phenylpropionate/cinnamic acid dioxygenase subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)216,47115
Polymers215,3266
Non-polymers1,1469
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 3-phenylpropionate/cinnamic acid dioxygenase subunit alpha


Mass: 51166.047 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: hcaE, digA, hcaA, hcaA1, phdC1, yfhU, b2538, JW2522
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0ABR5, 3-phenylpropanoate dioxygenase
#2: Protein 3-phenylpropionate/cinnamic acid dioxygenase subunit beta


Mass: 20609.248 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: hcaF, digB, hcaA2, hcaB, phdC2, yfhV, b2539, JW2523
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q47140, 3-phenylpropanoate dioxygenase
#3: Chemical ChemComp-FE2 / FE (II) ION


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: 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
#5: Chemical ChemComp-HCI / HYDROCINNAMIC ACID / 3PP / 3-PHENYLPROPIONIC ACID


Mass: 150.174 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H10O2 / Feature type: SUBJECT OF INVESTIGATION
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: complex of 3-phenylpropionate bound dioxygenase HcaE-HcaF
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152508 / Symmetry type: POINT
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415375
ELECTRON MICROSCOPYf_angle_d0.52420850
ELECTRON MICROSCOPYf_dihedral_angle_d11.4575610
ELECTRON MICROSCOPYf_chiral_restr0.0422169
ELECTRON MICROSCOPYf_plane_restr0.0042742

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