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- PDB-8ihq: Cryo-EM structure of ochratoxin A-detoxifying amidohydrolase ADH3 -

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Basic information

Entry
Database: PDB / ID: 8ihq
TitleCryo-EM structure of ochratoxin A-detoxifying amidohydrolase ADH3
ComponentsAmidohydrolase family protein
KeywordsHYDROLASE / amidohydrolase / octamer / ochratoxin A degradtion / cryo-EM structure
Function / homologyhydrolase activity, acting on carbon-nitrogen (but not peptide) bonds / Amidohydrolase family / Metal-dependent hydrolase, composite domain superfamily / Amidohydrolase-related / Metal-dependent hydrolase / Amidohydrolase family protein
Function and homology information
Biological speciesStenotrophomonas acidaminiphila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsDai, L.H. / Niu, D. / Huang, J.-W. / Li, X. / Shen, P.P. / Li, H. / Hu, Y.M. / Yang, Y. / Chen, C.-C. / Guo, R.-T.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: J Hazard Mater / Year: 2023
Title: Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase.
Authors: Longhai Dai / Du Niu / Jian-Wen Huang / Xian Li / Panpan Shen / Hao Li / Zhenzhen Xie / Jian Min / Yumei Hu / Yu Yang / Rey-Ting Guo / Chun-Chi Chen /
Abstract: Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing ...Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-β-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5-2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.
History
DepositionFeb 23, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)366,78724
Polymers365,7418
Non-polymers1,04716
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Amidohydrolase family protein / ADH3


Mass: 45717.617 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Stenotrophomonas acidaminiphila (bacteria)
Gene: H7691_12935 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A7L8TXW5
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn / 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: ADH3 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Stenotrophomonas acidaminiphila (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM Tris-HCL,pH 7.5
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
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

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141155 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00224608
ELECTRON MICROSCOPYf_angle_d0.44833376
ELECTRON MICROSCOPYf_dihedral_angle_d5.6043544
ELECTRON MICROSCOPYf_chiral_restr0.0433672
ELECTRON MICROSCOPYf_plane_restr0.0054448

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