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- PDB-6ycp: Crystal structure of GcoA F169V bound to o-vanillin -

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Basic information

Entry
Database: PDB / ID: 6ycp
TitleCrystal structure of GcoA F169V bound to o-vanillin
ComponentsAromatic O-demethylase, cytochrome P450 subunit
KeywordsOXIDOREDUCTASE / Aromatic catabolism / cytochrome P450 / lignin valorization / protein engineering
Function / homology
Function and homology information


Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor / : / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen / monooxygenase activity / iron ion binding / heme binding
Similarity search - Function
Cytochrome P450, B-class / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / 2-(hydroxymethyl)-6-methoxy-phenol / Aromatic O-demethylase, cytochrome P450 subunit
Similarity search - Component
Biological speciesAmycolatopsis sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsHinchen, D.J. / Mallinson, S.J.B. / Allen, M.D. / Ellis, E.S. / Beckham, G.T. / DuBois, J.L. / McGeehan, J.E.
Funding support United Kingdom, United States, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P011918/1 United Kingdom
Department of Energy (DOE, United States)DE-AC36-08GO28308 United States
CitationJournal: Jacs Au / Year: 2021
Title: Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.
Authors: Ellis, E.S. / Hinchen, D.J. / Bleem, A. / Bu, L. / Mallinson, S.J.B. / Allen, M.D. / Streit, B.R. / Machovina, M.M. / Doolin, Q.V. / Michener, W.E. / Johnson, C.W. / Knott, B.C. / Beckham, G. ...Authors: Ellis, E.S. / Hinchen, D.J. / Bleem, A. / Bu, L. / Mallinson, S.J.B. / Allen, M.D. / Streit, B.R. / Machovina, M.M. / Doolin, Q.V. / Michener, W.E. / Johnson, C.W. / Knott, B.C. / Beckham, G.T. / McGeehan, J.E. / DuBois, J.L.
History
DepositionMar 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 22, 2021Group: Data collection / Database references
Category: citation / database_2 ...citation / database_2 / diffrn_source / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site
Revision 1.2Jan 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / diffrn_source / pdbx_initial_refinement_model
Item: _diffrn_source.pdbx_synchrotron_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aromatic O-demethylase, cytochrome P450 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,1753
Polymers45,4051
Non-polymers7712
Water6,017334
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Correlates to 5NCB, which was confirmed as monomeric by SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1240 Å2
ΔGint-25 kcal/mol
Surface area16370 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.193, 104.193, 115.334
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-632-

HOH

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Components

#1: Protein Aromatic O-demethylase, cytochrome P450 subunit


Mass: 45404.734 Da / Num. of mol.: 1 / Mutation: F169V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Amycolatopsis sp. (strain ATCC 39116 / 75iv2) (bacteria)
Gene: gcoA / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): Rosetta II
References: UniProt: P0DPQ7, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one ...References: UniProt: P0DPQ7, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-OKE / 2-(hydroxymethyl)-6-methoxy-phenol / o-vanillin / Ortho-Vanillin


Mass: 154.163 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H10O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 334 / 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: 3.45 Å3/Da / Density % sol: 64.32 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / Details: Na Malonate, HEPES, o-vanillin

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.97955 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 22, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97955 Å / Relative weight: 1
ReflectionResolution: 1.799→47.48 Å / Num. obs: 59357 / % possible obs: 99.83 % / Redundancy: 2 % / Biso Wilson estimate: 31.45 Å2 / CC1/2: 1 / Net I/σ(I): 28.19
Reflection shellResolution: 1.799→1.864 Å / Rmerge(I) obs: 0.1377 / Mean I/σ(I) obs: 5.06 / Num. unique obs: 5809 / CC1/2: 0.947

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5NCB

5ncb


Resolution: 1.8→47.48 Å / SU ML: 0.1814 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 17.9772
RfactorNum. reflection% reflection
Rfree0.1831 2985 5.03 %
Rwork0.1617 --
obs0.1628 59353 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 38.27 Å2
Refinement stepCycle: LAST / Resolution: 1.8→47.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3162 0 54 334 3550
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00963318
X-RAY DIFFRACTIONf_angle_d1.13614550
X-RAY DIFFRACTIONf_chiral_restr0.0578480
X-RAY DIFFRACTIONf_plane_restr0.0086601
X-RAY DIFFRACTIONf_dihedral_angle_d15.91351945
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.830.25281410.22652617X-RAY DIFFRACTION99.07
1.83-1.860.28151410.21232650X-RAY DIFFRACTION99.96
1.86-1.890.24441200.21362654X-RAY DIFFRACTION100
1.89-1.930.25171450.20422630X-RAY DIFFRACTION100
1.93-1.970.24781330.19832670X-RAY DIFFRACTION100
1.97-2.010.21521570.18882628X-RAY DIFFRACTION100
2.01-2.060.17391460.18132643X-RAY DIFFRACTION100
2.06-2.110.21410.17392643X-RAY DIFFRACTION100
2.11-2.170.20531560.17312649X-RAY DIFFRACTION100
2.17-2.230.21971240.17492668X-RAY DIFFRACTION100
2.23-2.30.2011230.17342679X-RAY DIFFRACTION100
2.3-2.390.21191330.16672675X-RAY DIFFRACTION100
2.39-2.480.17891280.16192703X-RAY DIFFRACTION99.96
2.48-2.590.16881540.16712660X-RAY DIFFRACTION100
2.59-2.730.23541530.18472683X-RAY DIFFRACTION100
2.73-2.90.19711350.18052697X-RAY DIFFRACTION100
2.9-3.130.19881520.17242680X-RAY DIFFRACTION100
3.13-3.440.18321480.16032716X-RAY DIFFRACTION100
3.44-3.940.14231570.13012720X-RAY DIFFRACTION99.93
3.94-4.960.15191520.12182778X-RAY DIFFRACTION99.97
4.96-47.480.17131460.17142925X-RAY DIFFRACTION99.87

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