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- PDB-8yag: Cryo-electron microscopic structure of an amide hydrolase from Ps... -

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Basic information

Entry
Database: PDB / ID: 8yag
TitleCryo-electron microscopic structure of an amide hydrolase from Pseudoxanthomonas wuyuanensis
ComponentsImidazolonepropionase
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 / Imidazolonepropionase
Function and homology information
Biological speciesPseudoxanthomonas wuyuanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsDai, L.H. / Xu, Y.H. / Hu, Y.M. / Niu, D. / Yang, X.C. / Shen, P.P. / Li, X. / Xie, Z.Z. / Li, H. / Guo, R.-T. / Chen, C.-C.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Int J Biol Macromol / Year: 2024
Title: Functional characterization and structural basis of an efficient ochratoxin A-degrading amidohydrolase.
Authors: Yumei Hu / Longhai Dai / Yuhang Xu / Du Niu / Xuechun Yang / Zhenzhen Xie / Panpan Shen / Xian Li / Hao Li / Lilan Zhang / Jian Min / Rey-Ting Guo / Chun-Chi Chen /
Abstract: Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an ...Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.
History
DepositionFeb 9, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)348,39724
Polymers347,3508
Non-polymers1,04716
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Imidazolonepropionase / Amidohydrolase PwADH


Mass: 43418.758 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudoxanthomonas wuyuanensis (bacteria)
Gene: SAMN06296416_10433 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A286D6Z1
#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: PwADH / Type: COMPLEX
Details: amidohydrolase family protein [Pseudoxanthomonas wuyuanensis]
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Pseudoxanthomonas wuyuanensis (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

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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 875942 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00224424
ELECTRON MICROSCOPYf_angle_d0.46633128
ELECTRON MICROSCOPYf_dihedral_angle_d4.7513512
ELECTRON MICROSCOPYf_chiral_restr0.0443688
ELECTRON MICROSCOPYf_plane_restr0.0054400

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