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- PDB-8hfb: Evolved variant of quercetin 2,4-dioxygenase from Bacillus subtilis -

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Basic information

Entry
Database: PDB / ID: 8hfb
TitleEvolved variant of quercetin 2,4-dioxygenase from Bacillus subtilis
ComponentsQuercetin 2,3-dioxygenase
KeywordsOXIDOREDUCTASE / quercetinase
Function / homology
Function and homology information


quercetin 2,3-dioxygenase / quercetin 2,3-dioxygenase activity / metal ion binding
Similarity search - Function
: / Cupin 2, conserved barrel / Cupin domain / RmlC-like cupin domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
NICKEL (II) ION / Quercetin 2,3-dioxygenase
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.24 Å
AuthorsEom, H. / Song, W.J.
Funding support Korea, Republic Of, 2items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2021R1A5A1030054 Korea, Republic Of
National Research Foundation (NRF, Korea)2022R1A2C4001207 Korea, Republic Of
CitationJournal: J.Am.Chem.Soc. / Year: 2023
Title: Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis.
Authors: Eom, H. / Cao, Y. / Kim, H. / de Visser, S.P. / Song, W.J.
History
DepositionNov 10, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Quercetin 2,3-dioxygenase
B: Quercetin 2,3-dioxygenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,71811
Polymers75,1432
Non-polymers5759
Water3,369187
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Homo 2-mer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4320 Å2
ΔGint-50 kcal/mol
Surface area25420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.299, 125.211, 119.008
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Quercetin 2,3-dioxygenase / Quercetinase / Flavonol 2 / 4-dioxygenase


Mass: 37571.469 Da / Num. of mol.: 2 / Mutation: V221L, I231V, H239C, V269A, M286F, L290V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42106, quercetin 2,3-dioxygenase
#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 187 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.44 Å3/Da / Density % sol: 49.52 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 7.25 / Details: 18% PEG 3350, 0.1 M HEPES pH 7.25, 0.2 M CaCl2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PAL/PLS / Beamline: 5C (4A) / Wavelength: 1.00003 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Sep 26, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00003 Å / Relative weight: 1
ReflectionResolution: 2.21→50 Å / Num. obs: 62098 / % possible obs: 99.3 % / Redundancy: 6.2 % / Rmerge(I) obs: 0.108 / Χ2: 0.048 / Net I/σ(I): 4.7
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID% possible all
2.21-2.255.90.81131330.449199.3
2.25-2.296.10.72430900.444199.8
2.29-2.336.30.6731070.458199.8
2.33-2.386.50.61230880.463199.6
2.38-2.436.60.54431540.481199.7
2.43-2.496.70.47131330.502199.6
2.49-2.556.60.41530790.521199.6
2.55-2.626.60.34731260.549199.6
2.62-2.76.50.31531060.581199.3
2.7-2.786.40.25231290.653199.4
2.78-2.886.10.22131070.727199.4
2.88-35.60.18830860.795199.2
3-3.145.40.15330840.96198.4
3.14-3.36.20.1330991.018199.5
3.3-3.516.20.1130741.102198.9
3.51-3.7860.09131061.248198.5
3.78-4.165.80.07730681.355198.3
4.16-4.765.60.06330751.328198.2
4.76-65.60.05931041.13199.6
6-507.30.06631501.5271100

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Processing

Software
NameVersionClassification
HKL-2000data scaling
REFMAC5.8.0258refinement
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2H0V

2h0v


Resolution: 2.24→47.19 Å / Cor.coef. Fo:Fc: 0.949 / Cor.coef. Fo:Fc free: 0.93 / SU B: 7.427 / SU ML: 0.18 / Cross valid method: THROUGHOUT / ESU R: 0.349 / ESU R Free: 0.237 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.251 1626 5 %RANDOM
Rwork0.21332 ---
obs0.21517 30877 95.21 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 54.874 Å2
Baniso -1Baniso -2Baniso -3
1--0.68 Å2-0 Å2-0 Å2
2--1.87 Å20 Å2
3----1.19 Å2
Refinement stepCycle: 1 / Resolution: 2.24→47.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4920 0 26 187 5133
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0020.0135077
X-RAY DIFFRACTIONr_bond_other_d0.0010.0174577
X-RAY DIFFRACTIONr_angle_refined_deg1.1731.6476887
X-RAY DIFFRACTIONr_angle_other_deg1.0311.57110634
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.9355629
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.06122.293266
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.90815800
X-RAY DIFFRACTIONr_dihedral_angle_4_deg9.911528
X-RAY DIFFRACTIONr_chiral_restr0.0350.2631
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.025732
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021068
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.7715.912522
X-RAY DIFFRACTIONr_mcbond_other0.7715.912521
X-RAY DIFFRACTIONr_mcangle_it1.4168.8573149
X-RAY DIFFRACTIONr_mcangle_other1.4168.8583150
X-RAY DIFFRACTIONr_scbond_it0.555.9872554
X-RAY DIFFRACTIONr_scbond_other0.555.9872555
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.0238.9473739
X-RAY DIFFRACTIONr_long_range_B_refined5.071110.48219618
X-RAY DIFFRACTIONr_long_range_B_other5.056110.51219542
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.241→2.3 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.304 52 -
Rwork0.274 1114 -
obs--46.98 %

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