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- PDB-9l36: Cryo-electron microscopic structure of a novel amidohydrolase ADH... -

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Basic information

Entry
Database: PDB / ID: 9l36
TitleCryo-electron microscopic structure of a novel amidohydrolase ADH3 triple mutation
ComponentsAmidohydrolase
KeywordsHYDROLASE / amide hydrolase / Iron-binding / Ochratoxin A / ZN
Function / homology: / : / hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds / Amidohydrolase family / Metal-dependent hydrolase, composite domain superfamily / Amidohydrolase-related / Metal-dependent hydrolase / Chem-97U / Amidohydrolase
Function and homology information
Biological speciesStenotrophomonas sp. CW117 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsDai, L.H. / He, B.Y. / Hu, Y.M. / Xu, Y.H. / Huang, J.P. / Xie, Z.Z. / Li, H. / Niu, D. / 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 17, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 13, 2026Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 13, 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

Downloads & links

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Assembly

Deposited unit
A: Amidohydrolase
B: Amidohydrolase
C: Amidohydrolase
D: Amidohydrolase
E: Amidohydrolase
F: Amidohydrolase
G: Amidohydrolase
H: Amidohydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,26732
Polymers346,9908
Non-polymers4,27724
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
Amidohydrolase


Mass: 43373.777 Da / Num. of mol.: 8 / Mutation: S88R/I325A/D344N
Source method: isolated from a genetically manipulated source
Details: Sequence reference for strain 'Stenotrophomonas sp. CW117' is not available in UniProt at the time of biocuration. Current sequence reference is from UniProt id A0A0S1B1B6.
Source: (gene. exp.) Stenotrophomonas sp. CW117 (bacteria) / Gene: AOT14_24220 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0S1B1B6
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn
#3: Chemical
ChemComp-97U / (2~{S})-2-[[(3~{R})-5-chloranyl-3-methyl-8-oxidanyl-1-oxidanylidene-3,4-dihydroisochromen-7-yl]carbonylamino]-3-phenyl-propanoic acid


Mass: 403.813 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C20H18ClNO6 / Comment: toxin*YM
Has ligand of interestY
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: ADH3-S88R-I325A-D344N / Type: COMPLEX
Details: amidohydrolase family protein [Stenotrophomonas sp. CW117]
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Stenotrophomonas sp. CW117Stenotrophomonas sp. CW117
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCL,pH 8.0
SpecimenConc.: 5 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 170139 / Num. of class averages: 20 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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