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- PDB-6yci: Crystal structure of GcoA T296G bound to guaiacol -

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Basic information

Entry
Database: PDB / ID: 6yci
TitleCrystal structure of GcoA T296G bound to guaiacol
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 / Guaiacol / 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 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)45,9953
Polymers45,2551
Non-polymers7412
Water6,431357
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 area16260 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.065, 104.065, 116.731
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-635-

HOH

21A-934-

HOH

31A-950-

HOH

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Components

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


Mass: 45254.559 Da / Num. of mol.: 1 / Mutation: T296G
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-JZ3 / Guaiacol / 2-methoxyphenol


Mass: 124.137 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H8O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 357 / 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: 3.49 Å3/Da / Density % sol: 64.77 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / Details: Na Malonate, HEPES, guaiacol

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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 PILATUS3 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: 1.798→47.525 Å / Num. obs: 60018 / % possible obs: 99.89 % / Redundancy: 2 % / Biso Wilson estimate: 28.33 Å2 / CC1/2: 1 / Net I/σ(I): 26.18
Reflection shellResolution: 1.798→1.862 Å / Rmerge(I) obs: 0.1535 / Num. unique obs: 5887 / CC1/2: 0.957

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Processing

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

5ncb


Resolution: 1.8→47.52 Å / SU ML: 0.1739 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 21.7185
RfactorNum. reflection% reflection
Rfree0.1872 2915 4.86 %
Rwork0.1658 --
obs0.1668 60015 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 39.74 Å2
Refinement stepCycle: LAST / Resolution: 1.8→47.52 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3163 0 52 357 3572
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00923318
X-RAY DIFFRACTIONf_angle_d1.10424548
X-RAY DIFFRACTIONf_chiral_restr0.0543477
X-RAY DIFFRACTIONf_plane_restr0.0077602
X-RAY DIFFRACTIONf_dihedral_angle_d14.06941940
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.830.31360.26292636X-RAY DIFFRACTION98.26
1.83-1.860.23971380.22792679X-RAY DIFFRACTION99.93
1.86-1.890.24651270.21232683X-RAY DIFFRACTION99.86
1.89-1.930.24421340.20912679X-RAY DIFFRACTION100
1.93-1.970.22881300.19942689X-RAY DIFFRACTION99.96
1.97-2.010.19961370.19782682X-RAY DIFFRACTION99.96
2.01-2.060.29271470.19672683X-RAY DIFFRACTION99.96
2.06-2.110.22841320.19072682X-RAY DIFFRACTION99.96
2.11-2.170.2281310.18952721X-RAY DIFFRACTION100
2.17-2.230.20361350.18662675X-RAY DIFFRACTION100
2.23-2.30.23851420.17672706X-RAY DIFFRACTION99.93
2.3-2.380.20941370.17152692X-RAY DIFFRACTION100
2.38-2.480.21131650.16792700X-RAY DIFFRACTION99.97
2.48-2.590.1861300.16772697X-RAY DIFFRACTION100
2.59-2.730.20431560.18082715X-RAY DIFFRACTION100
2.73-2.90.18921490.18162720X-RAY DIFFRACTION100
2.9-3.120.20031470.18052726X-RAY DIFFRACTION100
3.12-3.440.17371370.16322750X-RAY DIFFRACTION100
3.44-3.940.15131140.13422806X-RAY DIFFRACTION100
3.94-4.960.1271530.12012801X-RAY DIFFRACTION99.97
4.96-47.520.18761380.17052978X-RAY DIFFRACTION99.94

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