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- PDB-6ycj: Crystal structure of GcoA T296S bound to guaiacol -

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Basic information

Entry
Database: PDB / ID: 6ycj
TitleCrystal structure of GcoA T296S 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: 1.64 Å
AuthorsMallinson, S.J.B. / Hinchen, D.J. / 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)46,1793
Polymers45,4391
Non-polymers7412
Water8,899494
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 area16040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)103.978, 103.978, 117.220
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-1072-

HOH

31A-1076-

HOH

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Components

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


Mass: 45438.750 Da / Num. of mol.: 1 / Mutation: T296S
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.: 494 / 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: 64.72 %
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: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Aug 3, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.64→62.29 Å / Num. obs: 78998 / % possible obs: 99.37 % / Redundancy: 12.8 % / Biso Wilson estimate: 21.69 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.06269 / Net I/σ(I): 19.15
Reflection shellResolution: 1.64→1.699 Å / Rmerge(I) obs: 1.261 / Num. unique obs: 7758 / CC1/2: 0.858

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
xia2data reduction
xia2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5NCB

5ncb


Resolution: 1.64→62.29 Å / SU ML: 0.2215 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 27.1621
RfactorNum. reflection% reflection
Rfree0.2118 3972 5.06 %
Rwork0.1926 --
obs0.1936 78529 99.37 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 27.85 Å2
Refinement stepCycle: LAST / Resolution: 1.64→62.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3115 0 52 494 3661
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00653328
X-RAY DIFFRACTIONf_angle_d0.91084580
X-RAY DIFFRACTIONf_chiral_restr0.0512484
X-RAY DIFFRACTIONf_plane_restr0.0059611
X-RAY DIFFRACTIONf_dihedral_angle_d3.73462632
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.64-1.660.37841190.39472603X-RAY DIFFRACTION97.28
1.66-1.680.3651490.37632576X-RAY DIFFRACTION97.99
1.68-1.70.38911460.36552588X-RAY DIFFRACTION97.75
1.7-1.730.36571490.35862574X-RAY DIFFRACTION98.7
1.73-1.750.33031310.32092611X-RAY DIFFRACTION98.74
1.75-1.780.31481470.30472615X-RAY DIFFRACTION99
1.78-1.810.36741410.2872615X-RAY DIFFRACTION99.35
1.81-1.830.26961450.27482623X-RAY DIFFRACTION99.14
1.83-1.870.30171350.26382634X-RAY DIFFRACTION99.11
1.87-1.90.30221520.26052601X-RAY DIFFRACTION99.14
1.9-1.940.26991360.24472641X-RAY DIFFRACTION99.39
1.94-1.980.29011440.2372628X-RAY DIFFRACTION99.5
1.98-2.020.2561300.2332679X-RAY DIFFRACTION99.61
2.02-2.070.27781360.22262662X-RAY DIFFRACTION99.68
2.07-2.120.25791250.21972657X-RAY DIFFRACTION99.61
2.12-2.180.28171380.2132669X-RAY DIFFRACTION99.96
2.18-2.240.27131280.20052666X-RAY DIFFRACTION99.82
2.24-2.310.23421570.19482650X-RAY DIFFRACTION99.61
2.31-2.390.20821500.18492634X-RAY DIFFRACTION99.75
2.39-2.490.2291510.19062696X-RAY DIFFRACTION99.96
2.49-2.60.2241580.18932675X-RAY DIFFRACTION99.82
2.6-2.740.2171250.22691X-RAY DIFFRACTION99.82
2.74-2.910.18811570.19492673X-RAY DIFFRACTION99.96
2.91-3.140.20031470.18412717X-RAY DIFFRACTION99.9
3.14-3.450.18761220.16862730X-RAY DIFFRACTION99.96
3.45-3.950.14211660.14082716X-RAY DIFFRACTION99.93
3.95-4.980.1341490.12762786X-RAY DIFFRACTION100
4.98-62.290.16481390.16232947X-RAY DIFFRACTION99.87

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