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- PDB-8jt7: Structure of arginine oxidase from Pseudomonas sp. TRU 7192 -

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Basic information

Entry
Database: PDB / ID: 8jt7
TitleStructure of arginine oxidase from Pseudomonas sp. TRU 7192
ComponentsAmine oxidoreductase
KeywordsOXIDOREDUCTASE
Function / homologyFLAVIN-ADENINE DINUCLEOTIDE
Function and homology information
Biological speciesPseudomonas sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.34 Å
AuthorsYamaguchi, H. / Numoto, N. / Suzuki, H. / Nishikawa, K. / Kamegawa, A. / Takahashi, K. / Sugiki, M. / Fujiyoshi, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biochem / Year: 2025
Title: Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography.
Authors: Hiroki Yamaguchi / Kazutoshi Takahashi / Nobutaka Numoto / Hiroshi Suzuki / Moemi Tatsumi / Akiko Kamegawa / Kouki Nishikawa / Yasuhisa Asano / Toshimi Mizukoshi / Hiroshi Miyano / Yoshinori ...Authors: Hiroki Yamaguchi / Kazutoshi Takahashi / Nobutaka Numoto / Hiroshi Suzuki / Moemi Tatsumi / Akiko Kamegawa / Kouki Nishikawa / Yasuhisa Asano / Toshimi Mizukoshi / Hiroshi Miyano / Yoshinori Fujiyoshi / Masayuki Sugiki /
Abstract: L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyses the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from ...L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyses the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.
History
DepositionJun 21, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 13, 2024Group: Data collection / Database references / Category: citation / em_admin
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Revision 1.3Jan 15, 2025Group: Data collection / Database references / Category: citation / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Amine oxidoreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,1792
Polymers67,3931
Non-polymers7861
Water52229
1
A: Amine oxidoreductase
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)545,43016
Polymers539,1458
Non-polymers6,2848
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation7
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
2generate(6.123234E-17, -1), (1, 6.123234E-17), (1)203.51997
3generate(-1, -1.2246468E-16), (1.2246468E-16, -1), (1)203.51997, 203.51997
4generate(-1.8369702E-16, 1), (-1, -1.8369702E-16), (1)1.42108547E-14, 203.51997
5generate(1), (-1, -1.2246468E-16), (1.2246468E-16, -1)203.51997, 203.51997
6generate(2.22044605E-16, 1, 8.65956056E-17), (1, 2.22044605E-16, -8.65956056E-17), (-8.65956056E-17, 8.65956056E-17, -1)-7.10542736E-14, -4.26325641E-14, 203.51997
7generate(2.22044605E-16, -1, 8.65956056E-17), (-1, 2.22044605E-16, 8.65956056E-17), (-8.65956056E-17, -8.65956056E-17, -1)203.51997, 203.51997, 203.51997
8generate(-1, 1.2246468E-16), (1), (-1.2246468E-16, -1)203.51997, 203.51997

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Components

#1: Protein Amine oxidoreductase


Mass: 67393.164 Da / Num. of mol.: 1 / Fragment: initiation methionine (1A), his-tags (2-7A)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas sp. (bacteria) / Strain: TPU 7192 / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 29 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: homooctamer of arginine oxidase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.54 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas sp. (bacteria) / Strain: TPU 7192
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
250 mMsodium chlorideNaCl1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA KF80 / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 3.4 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 69.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5035
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2SerialEM3.8.9image acquisition
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
19REFMACmodel refinement
20Servalcat0.4.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 254111 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model

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