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- PDB-6ycn: Crystal structure of GcoA F169A bound to o-vanillin -

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Basic information

Entry
Database: PDB / ID: 6ycn
TitleCrystal structure of GcoA F169A 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 / catabolic process / 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: 1.83 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 29, 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)46,1473
Polymers45,3771
Non-polymers7712
Water5,891327
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 area1230 Å2
ΔGint-25 kcal/mol
Surface area16330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.328, 104.328, 115.025
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-625-

HOH

21A-914-

HOH

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Components

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


Mass: 45376.680 Da / Num. of mol.: 1 / Mutation: F169A
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


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


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


Mass: 18.015 Da / Num. of mol.: 327 / 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: 64.34 %
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: 1.83→47.51 Å / Num. obs: 56447 / % possible obs: 99.94 % / Redundancy: 2 % / Biso Wilson estimate: 33.57 Å2 / CC1/2: 0.943 / Rmerge(I) obs: 0.012 / Rrim(I) all: 0.01806 / Net I/σ(I): 31.96
Reflection shellResolution: 1.83→1.895 Å / Rmerge(I) obs: 0.1503 / Mean I/σ(I) obs: 5.17 / Num. unique obs: 5530 / CC1/2: 0.943

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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: 1.83→47.51 Å / SU ML: 0.1555 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 17.1663
RfactorNum. reflection% reflection
Rfree0.179 2899 5.13 %
Rwork0.1563 --
obs0.1575 56447 99.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 40.92 Å2
Refinement stepCycle: LAST / Resolution: 1.83→47.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3160 0 54 327 3541
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01063316
X-RAY DIFFRACTIONf_angle_d1.01354547
X-RAY DIFFRACTIONf_chiral_restr0.0557479
X-RAY DIFFRACTIONf_plane_restr0.0077601
X-RAY DIFFRACTIONf_dihedral_angle_d15.00141944
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.83-1.860.23711340.20982435X-RAY DIFFRACTION98.35
1.86-1.890.22411530.20052518X-RAY DIFFRACTION100
1.89-1.930.23591360.19652505X-RAY DIFFRACTION100
1.93-1.960.23761220.19152534X-RAY DIFFRACTION100
1.96-20.25721380.18222496X-RAY DIFFRACTION100
2-2.050.20471310.1822550X-RAY DIFFRACTION99.96
2.05-2.090.22991520.17172482X-RAY DIFFRACTION100
2.09-2.150.17871510.16292507X-RAY DIFFRACTION100
2.15-2.20.19391420.16232526X-RAY DIFFRACTION100
2.2-2.270.1681370.15682528X-RAY DIFFRACTION100
2.27-2.340.16281340.16112545X-RAY DIFFRACTION100
2.34-2.430.17991390.15622550X-RAY DIFFRACTION99.96
2.43-2.520.16971200.1612523X-RAY DIFFRACTION100
2.52-2.640.18991310.16362568X-RAY DIFFRACTION100
2.64-2.780.20911430.17532551X-RAY DIFFRACTION100
2.78-2.950.21461200.17792576X-RAY DIFFRACTION100
2.95-3.180.19241380.1722567X-RAY DIFFRACTION100
3.18-3.50.18671370.15252583X-RAY DIFFRACTION100
3.5-4.010.14261490.12712598X-RAY DIFFRACTION100
4.01-5.050.14231290.11982650X-RAY DIFFRACTION100
5.05-47.510.17961630.17042766X-RAY DIFFRACTION99.83

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