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- EMDB-62782: Cryo-electron microscopic structure of a novel amidohydrolase ADH... -

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Basic information

Entry
Database: EMDB / ID: EMD-62782
TitleCryo-electron microscopic structure of a novel amidohydrolase ADH3 triple mutation
Map data
Sample
  • Complex: ADH3-S88R-I325A-D344N
    • Protein or peptide: Amidohydrolase
  • Ligand: ZINC ION
  • Ligand: (2~{S})-2-[[(3~{R})-5-chloranyl-3-methyl-8-oxidanyl-1-oxidanylidene-3,4-dihydroisochromen-7-yl]carbonylamino]-3-phenyl-propanoic acid
Keywordsamide hydrolase / Iron-binding / Ochratoxin A / ZN / HYDROLASE
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 / Amidohydrolase
Function and homology information
Biological speciesStenotrophomonas sp. CW117Stenotrophomonas sp. CW117 (bacteria) / Stenotrophomonas sp. CW117 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsDai LH / He BY / Hu YM / Xu YH / Huang JP / Xie ZZ / Li H / Niu D / Guo R-T / Chen C-C
Funding support1 items
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, 2024-
Header (metadata) releaseMay 13, 2026-
Map releaseMay 13, 2026-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62782.png

Downloads & links

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Map

FileDownload / File: emd_62782.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-1.4440306 - 2.440905
Average (Standard dev.)0.0009291736 (±0.069423534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 334.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62782_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_62782_half_map_2.map
Projections & Slices
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Sample components

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Entire : ADH3-S88R-I325A-D344N

EntireName: ADH3-S88R-I325A-D344N
Components
  • Complex: ADH3-S88R-I325A-D344N
    • Protein or peptide: Amidohydrolase
  • Ligand: ZINC ION
  • Ligand: (2~{S})-2-[[(3~{R})-5-chloranyl-3-methyl-8-oxidanyl-1-oxidanylidene-3,4-dihydroisochromen-7-yl]carbonylamino]-3-phenyl-propanoic acid

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Supramolecule #1: ADH3-S88R-I325A-D344N

SupramoleculeName: ADH3-S88R-I325A-D344N / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: amidohydrolase family protein [Stenotrophomonas sp. CW117]
Source (natural)Organism: Stenotrophomonas sp. CW117Stenotrophomonas sp. CW117 (bacteria)

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Macromolecule #1: Amidohydrolase

MacromoleculeName: Amidohydrolase / type: protein_or_peptide / ID: 1
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.
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Stenotrophomonas sp. CW117 (bacteria)
Molecular weightTheoretical: 43.373777 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PVAVQCGRLF DARSGQLKGP HTLLVADGRI RQVLPGTGAD AAGARVVDLG DKVCLPGWTD LHVHLGRQSS PQSYSEDFRL DPVDHAFRA VGYAEKTLMA GFTSVRDLGG EVSPHLRDAI NQGLVRGPRI FAAGKSIATT GGHADPTNGW NERLAHLVGA P GPAEGVVN ...String:
PVAVQCGRLF DARSGQLKGP HTLLVADGRI RQVLPGTGAD AAGARVVDLG DKVCLPGWTD LHVHLGRQSS PQSYSEDFRL DPVDHAFRA VGYAEKTLMA GFTSVRDLGG EVSPHLRDAI NQGLVRGPRI FAAGKSIATT GGHADPTNGW NERLAHLVGA P GPAEGVVN SVDEARQAVR QRYKEGSDLI (KCX)ITATGGVLS YARSGDAPQF TVDEIKAVVD TARDYGFRVA AHAHGTEG M KRAVQAGVTS IEHGTYMDDE VMRLMKQHGT WYVPTFYAGR FVTEKAAIDG YFPEVVRPKA ARIGALASQT AAKAYRNGV RIAFGTNQGV GPHGDNAREF VYMVEAGIPA AYALQAATVH AAQVLGVDDQ GVLEPGKRAD VIALAGNPLE DINAVLDVRF VMKDGVIYK Q

UniProtKB: Amidohydrolase

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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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Macromolecule #3: (2~{S})-2-[[(3~{R})-5-chloranyl-3-methyl-8-oxidanyl-1-oxidanylide...

MacromoleculeName: (2~{S})-2-[[(3~{R})-5-chloranyl-3-methyl-8-oxidanyl-1-oxidanylidene-3,4-dihydroisochromen-7-yl]carbonylamino]-3-phenyl-propanoic acid
type: ligand / ID: 3 / Number of copies: 8 / Formula: 97U
Molecular weightTheoretical: 403.813 Da
Chemical component information

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 / toxin*YM

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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

Concentration5 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCL,pH 8.0
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI MORGAGNI
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

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 20 / Resolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 170139
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9l36:
Cryo-electron microscopic structure of a novel amidohydrolase ADH3 triple mutation

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