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- EMDB-35452: Cryo-EM structure of ochratoxin A-detoxifying amidohydrolase ADH3 -

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Basic information

Entry
Database: EMDB / ID: EMD-35452
TitleCryo-EM structure of ochratoxin A-detoxifying amidohydrolase ADH3
Map data
Sample
  • Complex: ADH3
    • Protein or peptide: Amidohydrolase family protein
  • Ligand: ZINC ION
Keywordsamidohydrolase / octamer / ochratoxin A degradtion / cryo-EM structure / HYDROLASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsDai LH / Niu D / Huang J-W / Li X / Shen PP / Li H / Hu YM / Yang Y / Chen C-C / Guo R-T
Funding support China, 1 items
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, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_35452.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 360 pix.
= 306. Å
0.85 Å/pix.
x 360 pix.
= 306. Å
0.85 Å/pix.
x 360 pix.
= 306. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.99859303 - 1.7493086
Average (Standard dev.)0.0011987791 (±0.048941795)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35452_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_35452_half_map_2.map
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Sample components

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Entire : ADH3

EntireName: ADH3
Components
  • Complex: ADH3
    • Protein or peptide: Amidohydrolase family protein
  • Ligand: ZINC ION

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Supramolecule #1: ADH3

SupramoleculeName: ADH3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Stenotrophomonas acidaminiphila (bacteria)

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Macromolecule #1: Amidohydrolase family protein

MacromoleculeName: Amidohydrolase family protein / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Stenotrophomonas acidaminiphila (bacteria)
Molecular weightTheoretical: 45.717617 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPIRRRFASL LLLACAPAWA EPVAVQCGRL FDARSGQLKG PHTLLVADGR IRQVLPGTGA DAAGARVVDL GDKVCLPGWT DLHVHLGSQ SSPQSYSEDF RLDPVDHAFR AVGYAEKTLM AGFTSVRDLG GEVSPHLRDA INQGLVRGPR IFAAGKSIAT T GGHADPTN ...String:
MPIRRRFASL LLLACAPAWA EPVAVQCGRL FDARSGQLKG PHTLLVADGR IRQVLPGTGA DAAGARVVDL GDKVCLPGWT DLHVHLGSQ SSPQSYSEDF RLDPVDHAFR AVGYAEKTLM AGFTSVRDLG GEVSPHLRDA INQGLVRGPR IFAAGKSIAT T GGHADPTN GWNERLAHLV GAPGPAEGVV NSVDEARQAV RQRYKEGSDL I(KCX)ITATGGVL SYARSGDAPQ FTVDEIKA V VDTARDYGFR VAAHAHGTEG MKRAVQAGVT SIEHGTYMDD EVMRLMKQHG TWYVPTFYAG RFVTEKAAID GYFPEVVRP KAARIGALIS QTAAKAYRNG VRIAFGTDQG VGPHGDNARE FVYMVEAGIP AAYALQAATV HAAQVLGVDD QGVLEPGKRA DVIALAGNP LEDINAVLDV RFVMKDGVIY KQ

UniProtKB: Amidohydrolase family protein

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 16 / Formula: ZN
Molecular weightTheoretical: 65.409 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 / Details: 20 mM Tris-HCL,pH 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 141155
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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