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- PDB-9l6p: Cryo-electron microscopic structure of a highly efficient ochrato... -

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Basic information

Entry
Database: PDB / ID: 9l6p
TitleCryo-electron microscopic structure of a highly efficient ochratoxin detoxification enzyme LlADH
ComponentsLlADH-WT
KeywordsHYDROLASE / amide hydrolase / Iron-binding / Ochratoxin A / ZN / TOXIN
Biological speciesNovilysobacter luteus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsDai, L.H. / Xu, Y.H. / Hu, Y.M. / Niu, D. / He, B.Y. / Huang, J.P. / Xie, Z.Z. / Li, H. / Guo, R.-T. / Chen, C.-C.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Int J Biol Macromol / Year: 2026
Title: Structure-guided protein engineering and immobilization of an amidohydrolase for efficient ochratoxin A detoxification.
Authors: Yumei Hu / Yuhang Xu / Biyu He / Du Niu / Xuechun Yang / Jiaping Huang / Zhenzhen Xie / Panpan Shen / Xian Li / Maosen Bai / Ziwei Liu / Hao Li / Yunyun Yang / Jian-Wen Huang / Chun-Chi Chen ...Authors: Yumei Hu / Yuhang Xu / Biyu He / Du Niu / Xuechun Yang / Jiaping Huang / Zhenzhen Xie / Panpan Shen / Xian Li / Maosen Bai / Ziwei Liu / Hao Li / Yunyun Yang / Jian-Wen Huang / Chun-Chi Chen / Rey-Ting Guo / Longhai Dai /
Abstract: Ochratoxin A (OTA) is a pervasive and significant mycotoxin that poses serious health risks to humans and animals. The development of efficient biocatalysts for the enzymatic detoxification of OTA is ...Ochratoxin A (OTA) is a pervasive and significant mycotoxin that poses serious health risks to humans and animals. The development of efficient biocatalysts for the enzymatic detoxification of OTA is of great importance. In this study, we enhanced the OTA-degrading activity of three amidohydrolases (ADHs) by up to ninefold. This improvement was achieved by reducing steric hindrance in the binding pocket and fine-tuning the hydrophilic interactions between the enzyme and substrate. The most efficient variant, PwADH/DM, was immobilized onto magnetic FeO nanoparticles functionalized by the co-deposition of dopamine and polyethyleneimine. Under optimal conditions, this immobilization process achieved a high immobilization efficiency (85.4%) and activity recovery (76.9%). The immobilized enzyme exhibited enhanced pH stability and thermostability, along with good storage stability, reusability, and recyclability. More importantly, the immobilized enzyme completely degraded 100 ng/mL OTA in contaminated milk without affecting the milk's properties. These findings expand our understanding of the molecular mechanisms governing substrate binding and catalysis in OTA-degrading ADHs. Furthermore, they provide a blueprint for enzyme-based OTA decontamination during food processing.
History
DepositionDec 24, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 6, 2026Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 6, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: LlADH-WT
B: LlADH-WT
C: LlADH-WT
D: LlADH-WT
E: LlADH-WT
F: LlADH-WT
G: LlADH-WT
H: LlADH-WT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,47724
Polymers356,4318
Non-polymers1,04716
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
LlADH-WT


Mass: 44553.848 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Novilysobacter luteus (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
Has protein modificationY

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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: LL-WT / Type: COMPLEX / Details: amidohydrolase family protein / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Novilysobacter luteus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCL,pH 8.0
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI MORGAGNI
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125111 / Num. of class averages: 30 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023015
ELECTRON MICROSCOPYf_angle_d0.4044098
ELECTRON MICROSCOPYf_dihedral_angle_d5.589440
ELECTRON MICROSCOPYf_chiral_restr0.042457
ELECTRON MICROSCOPYf_plane_restr0.004544

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