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Entry
Database: PDB / ID: 8yak
TitleCryo-EM structure and rational engineering of a novel efficient ochratoxin A-detoxifying amidohydrolase
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 / Chem-97U / Imidazolonepropionase
Function and homology information
Biological speciesPseudoxanthomonas wuyuanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 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 1.0Dec 18, 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)352,08432
Polymers347,8068
Non-polymers4,27724
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Imidazolonepropionase / Amidohydrolase family protein


Mass: 43475.809 Da / Num. of mol.: 8 / Mutation: G88D, D344N
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
#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 / Feature type: SUBJECT OF INVESTIGATION / 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: PwADH-G88D-D344N / 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.: 1.5 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: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118429 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.37 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033087
ELECTRON MICROSCOPYf_angle_d0.634190
ELECTRON MICROSCOPYf_dihedral_angle_d4.932450
ELECTRON MICROSCOPYf_chiral_restr0.059464
ELECTRON MICROSCOPYf_plane_restr0.004556

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