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- PDB-7egn: Crystal structure of the P450 BM3 heme domain mutant F87A in comp... -

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Basic information

Entry
Database: PDB / ID: 7egn
TitleCrystal structure of the P450 BM3 heme domain mutant F87A in complex with N-imidazolyl-hexanoyl-L-phenylalanine and hydroxylamine
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / Dual-functional small molecule / P450 heme domain / Complex
Function / homology
Function and homology information


NADPH-hemoprotein reductase / NADPH-hemoprotein reductase activity / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen / unspecific monooxygenase / aromatase activity / FMN binding / flavin adenine dinucleotide binding / iron ion binding / heme binding / identical protein binding / cytosol
Similarity search - Function
Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / Oxidoreductase FAD/NAD(P)-binding ...Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Flavoprotein-like superfamily / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / HYDROXYAMINE / Chem-IC6 / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsJiang, Y. / Dong, S. / Feng, Y. / Cong, Z.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21778060 China
National Natural Science Foundation of China (NSFC)21977104 China
CitationJournal: Acs Catalysis / Year: 2021
Title: H-Bonding Networks Dictate the Molecular Mechanism of H2O2 Activation by P450
Authors: Zhang, X. / Jiang, Y. / Chen, Q. / Dong, S. / Feng, Y. / Cong, Z. / Shaik, S. / Wang, B.
History
DepositionMar 24, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional cytochrome P450/NADPH--P450 reductase
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,7559
Polymers106,7062
Non-polymers2,0507
Water2,666148
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4330 Å2
ΔGint-49 kcal/mol
Surface area35400 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.670, 147.557, 64.324
Angle α, β, γ (deg.)90.000, 99.980, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 14 or (resid 15...
21(chain B and (resid 3 through 8 or (resid 9...

NCS domain segments:

Ens-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS

Dom-IDComponent-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LEULEU(chain A and (resid 3 through 14 or (resid 15...AA3 - 145 - 16
12LYSLYS(chain A and (resid 3 through 14 or (resid 15...AA1517
13LEULEU(chain A and (resid 3 through 14 or (resid 15...AA3 - 4555 - 457
14LEULEU(chain A and (resid 3 through 14 or (resid 15...AA3 - 4555 - 457
15LEULEU(chain A and (resid 3 through 14 or (resid 15...AA3 - 4555 - 457
21PROPRO(chain B and (resid 3 through 8 or (resid 9...BB3 - 85 - 10
22LYSLYS(chain B and (resid 3 through 8 or (resid 9...BB911
23LEULEU(chain B and (resid 3 through 8 or (resid 9...BB3 - 4555 - 457

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Bifunctional cytochrome P450/NADPH--P450 reductase / Cytochrome P450(BM-3) / Cytochrome P450BM-3 / Fatty acid monooxygenase / Flavocytochrome P450 BM3


Mass: 53352.758 Da / Num. of mol.: 2 / Mutation: F87A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (strain ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / VKM B-512) (bacteria)
Gene: cyp102A1, cyp102, BG04_163 / Plasmid: PET-28a / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase

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Non-polymers , 5 types, 155 molecules

#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-IC6 / (2S)-2-(6-imidazol-1-ylhexanoylamino)-3-phenyl-propanoic acid


Mass: 329.394 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H23N3O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-HOA / HYDROXYAMINE


Mass: 33.030 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: H3NO / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 148 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.57 Å3/Da / Density % sol: 52.14 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.1M Tris, 0.2M MgCl2, 27% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9789 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jan 30, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9789 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 29471 / % possible obs: 99.8 % / Redundancy: 3.4 % / Rmerge(I) obs: 0.168 / Rpim(I) all: 0.108 / Rrim(I) all: 0.2 / Χ2: 0.875 / Net I/σ(I): 4.3 / Num. measured all: 101089
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.7-2.753.20.6714600.6190.4510.810.637100
2.75-2.83.50.54215070.5270.3390.6410.71399.9
2.8-2.853.30.47814360.7310.3080.570.7699.9
2.85-2.913.30.45614800.7840.2940.5440.667100
2.91-2.973.60.42914340.8440.2620.5040.6599.9
2.97-3.043.60.35115220.8810.2130.4110.67100
3.04-3.123.50.32214310.8930.1980.3790.73399.9
3.12-3.23.50.2914860.9050.180.3420.74699.9
3.2-3.33.50.30114710.8260.1870.3551.06599.8
3.3-3.43.40.25514640.8710.1620.3030.759100
3.4-3.523.20.26314610.8460.1790.321.42699.2
3.52-3.663.40.21114810.9540.1380.2531.10599.5
3.66-3.833.30.17414550.9310.1130.2080.98499.7
3.83-4.033.50.18514720.8510.1180.221.3299.6
4.03-4.293.60.12514960.9710.0760.1460.87999.9
4.29-4.623.50.10814630.9760.0670.1280.89199.9
4.62-5.083.40.10314700.9780.0660.1220.86299.7
5.08-5.813.40.11214930.970.0720.1330.81999.7
5.81-7.323.60.09814990.9820.0610.1160.73399.9
7.32-503.30.09614900.980.0640.1161.11299

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
HKL-3000data scaling
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5B2V

5b2v


Resolution: 2.7→39.43 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.37 / Phase error: 26.38 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2446 2000 7.26 %
Rwork0.2002 25535 -
obs0.2035 27535 92.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 90.9 Å2 / Biso mean: 38.7778 Å2 / Biso min: 16.04 Å2
Refinement stepCycle: final / Resolution: 2.7→39.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7075 0 144 148 7367
Biso mean--42.77 35.28 -
Num. residues----900
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2738X-RAY DIFFRACTION5.388TORSIONAL
12B2738X-RAY DIFFRACTION5.388TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7-2.770.3132960.23331229132564
2.77-2.840.29151150.22011472158775
2.84-2.920.29981280.23391634176282
2.92-3.020.29161390.24141773191293
3.02-3.130.33971490.23961900204997
3.13-3.250.30131530.23861951210498
3.25-3.40.29241490.222719192068100
3.4-3.580.27611540.2261952210698
3.58-3.80.27481500.211921207199
3.8-4.10.20941520.18991940209299
4.1-4.510.21091540.161619662120100
4.51-5.160.17241520.160319472099100
5.16-6.490.25221550.204519842139100
6.5-39.430.19071540.17631947210198

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