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- PDB-6f97: Crystal structure of the V465T mutant of 5-(Hydroxymethyl)furfura... -

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Basic information

Entry
Database: PDB / ID: 6f97
TitleCrystal structure of the V465T mutant of 5-(Hydroxymethyl)furfural Oxidase (HMFO)
Components5-(hydroxymethyl)furfural oxidase
KeywordsFLAVOPROTEIN / sec-thiol oxidation / alcohol oxidase / kinetic resolution / biocatalysis / enzyme engineering
Function / homology
Function and homology information


5-(hydroxymethyl)furfural oxidase / Oxidoreductases; Acting on a sulfur group of donors; With oxygen as acceptor / oxidoreductase activity, acting on a sulfur group of donors, oxygen as acceptor / oxidoreductase activity, acting on the CH-OH group of donors, oxygen as acceptor / FAD binding
Similarity search - Function
Cholesterol Oxidase; domain 2 - #40 / Cholesterol Oxidase; domain 2 / Glucose-methanol-choline oxidoreductase / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich ...Cholesterol Oxidase; domain 2 - #40 / Cholesterol Oxidase; domain 2 / Glucose-methanol-choline oxidoreductase / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich / FAD/NAD(P)-binding domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / 5-(hydroxymethyl)furfural oxidase
Similarity search - Component
Biological speciesMethylovorus sp.
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsPickl, M. / Swoboda, A. / Romero, E. / Winkler, C.K. / Binda, C. / Mattevi, A. / Faber, K. / Fraaije, M.W.
CitationJournal: Angew. Chem. Int. Ed. Engl. / Year: 2018
Title: Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase.
Authors: Pickl, M. / Swoboda, A. / Romero, E. / Winkler, C.K. / Binda, C. / Mattevi, A. / Faber, K. / Fraaije, M.W.
History
DepositionDec 14, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2018Group: Database references / Category: citation / citation_author / Item: _citation.title / _citation_author.name
Revision 1.2Mar 14, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 5-(hydroxymethyl)furfural oxidase
B: 5-(hydroxymethyl)furfural oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,7274
Polymers114,1562
Non-polymers1,5712
Water6,107339
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4200 Å2
ΔGint-24 kcal/mol
Surface area36580 Å2
MethodPISA
Unit cell
Length a, b, c (Å)51.189, 122.004, 72.698
Angle α, β, γ (deg.)90.00, 91.63, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein 5-(hydroxymethyl)furfural oxidase / 5-hydroxymethylfurfural oxidase / HMFO / Thiol oxidase


Mass: 57078.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The sequence contains the V465T mutation (with respect to the wild type sequence) which was intentionally introduced by site-directed mutagenesis.
Source: (gene. exp.) Methylovorus sp. (strain MP688) (bacteria)
Strain: MP688 / Gene: MPQ_0130 / Production host: Escherichia coli (E. coli)
References: UniProt: E4QP00, 5-(hydroxymethyl)furfural oxidase, Oxidoreductases; Acting on a sulfur group of donors; With oxygen as acceptor
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 339 / Source method: isolated from a natural source / Formula: H2O

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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: 1.99 Å3/Da / Density % sol: 38.12 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.6 / Details: 20% w/v PEG3350 and 200 mM magnesium formate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.988 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Sep 15, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.988 Å / Relative weight: 1
ReflectionResolution: 1.9→72.67 Å / Num. obs: 69743 / % possible obs: 98.4 % / Redundancy: 3 % / CC1/2: 0.998 / Rmerge(I) obs: 0.053 / Net I/σ(I): 9.3
Reflection shellResolution: 1.9→1.94 Å / Redundancy: 3 % / Rmerge(I) obs: 1.079 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 4440 / CC1/2: 0.507 / % possible all: 99.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0189refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4UDP

4udp


Resolution: 1.9→72.67 Å / Cor.coef. Fo:Fc: 0.975 / Cor.coef. Fo:Fc free: 0.953 / SU B: 6.205 / SU ML: 0.162 / Cross valid method: THROUGHOUT / ESU R: 0.162 / ESU R Free: 0.156 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2311 3334 4.8 %RANDOM
Rwork0.17162 ---
obs0.17442 66372 99.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 44.012 Å2
Baniso -1Baniso -2Baniso -3
1-3.71 Å20 Å20.23 Å2
2---0.18 Å20 Å2
3----3.54 Å2
Refinement stepCycle: 1 / Resolution: 1.9→72.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7644 0 106 339 8089
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0197952
X-RAY DIFFRACTIONr_bond_other_d0.0020.027338
X-RAY DIFFRACTIONr_angle_refined_deg1.811.9810877
X-RAY DIFFRACTIONr_angle_other_deg1.064316931
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.1251004
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.71222.933341
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.443151169
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.0761571
X-RAY DIFFRACTIONr_chiral_restr0.1060.21215
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0218943
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021640
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_it3.4094.2094025
X-RAY DIFFRACTIONr_mcbond_other3.4084.2094024
X-RAY DIFFRACTIONr_mcangle_it4.7026.2925020
X-RAY DIFFRACTIONr_mcangle_other4.7026.2925021
X-RAY DIFFRACTIONr_scbond_it4.0844.6373927
X-RAY DIFFRACTIONr_scbond_other4.0834.6373928
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.1046.7795856
X-RAY DIFFRACTIONr_long_range_B_refined7.76649.338748
X-RAY DIFFRACTIONr_long_range_B_other7.74949.2198689
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.404 232 -
Rwork0.369 4855 -
obs--99.51 %

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