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- PDB-3cbd: Directed Evolution of cytochrome P450 BM3, to octane monoxygenase... -

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Entry
Database: PDB / ID: 3cbd
TitleDirected Evolution of cytochrome P450 BM3, to octane monoxygenase 139-3
ComponentsBifunctional P-450/NADPH-P450 reductase
KeywordsOXIDOREDUCTASE / Cytochrome P450 / Hemeprotein 139-3 / Electron transport / FAD / Flavoprotein / FMN / Iron / Membrane / Metal-binding / Monooxygenase / Multifunctional enzyme / NADP / Transport
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 / metabolic process / response to hormone / FMN binding / flavin adenine dinucleotide binding / iron ion binding ...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 / metabolic process / response to hormone / 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 / Cytochrome p450 / Cytochrome P450 / 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. ...Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Cytochrome p450 / Cytochrome P450 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
N-PALMITOYLGLYCINE / PROTOPORPHYRIN IX CONTAINING FE / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.65 Å
AuthorsMeharenna, Y.T. / Li, H. / Poulos, T.L.
CitationJournal: To be Published
Title: Directed Evolution of cytochrome P450 BM3, to octane monoxygenase 139-3
Authors: Fasan, R. / Meharenna, Y.T. / Li, H. / Snow, C.D. / Poulos, T.L. / Arnold, F.H.
History
DepositionFeb 21, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bifunctional P-450/NADPH-P450 reductase
B: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,0856
Polymers104,2252
Non-polymers1,8604
Water5,062281
1
A: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,0423
Polymers52,1121
Non-polymers9302
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,0423
Polymers52,1121
Non-polymers9302
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)61.781, 127.346, 183.342
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Bifunctional P-450/NADPH-P450 reductase / Cytochrome P450(BM-3) / P450BM-3


Mass: 52112.418 Da / Num. of mol.: 2 / Fragment: Cytochrome P450 heme domain, residues 2-456
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (bacteria) / Gene: CYP102 / Plasmid: PPROEX-1 / Production host: Escherichia coli (E. coli) / Strain (production host): DH5alpha / References: UniProt: P14779, unspecific monooxygenase
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-140 / N-PALMITOYLGLYCINE / N-HEXADECANOYLGLYCINE


Mass: 313.475 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H35NO3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 281 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

CrystalDensity Matthews: 3.46 Å3/Da / Density % sol: 64.45 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 20% PEG 20,000, 50mM LiBr,100mM MOPS, pH 7.0, VAPOR DIFFUSION, HANGING DROP, temperature 298.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 1.5418 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Dec 19, 2003 / Details: mirrors
RadiationMonochromator: Double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.65→50 Å / Num. all: 42077 / Num. obs: 41994 / % possible obs: 97.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 3.6 % / Biso Wilson estimate: 66.8 Å2 / Rmerge(I) obs: 0.2 / Rsym value: 0.2
Reflection shellResolution: 2.65→2.74 Å / Redundancy: 1.6 % / Rmerge(I) obs: 0.124 / Rsym value: 0.124 / % possible all: 97.9

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data collection
HKL-2000data reduction
SCALEPACKdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1FAG

1fag


Resolution: 2.65→47.53 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 1623681.86 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.258 4241 10.1 %RANDOM
Rwork0.211 ---
all0.33 42077 --
obs0.211 41994 97.7 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 34.077 Å2 / ksol: 0.299968 e/Å3
Displacement parametersBiso mean: 59 Å2
Baniso -1Baniso -2Baniso -3
1-5.57 Å20 Å20 Å2
2--10.44 Å20 Å2
3----16.02 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.42 Å0.35 Å
Luzzati d res low-5 Å
Luzzati sigma a0.45 Å0.42 Å
Refinement stepCycle: LAST / Resolution: 2.65→47.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7334 0 130 281 7745
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d20.9
X-RAY DIFFRACTIONc_improper_angle_d1.04
LS refinement shellResolution: 2.65→2.74 Å / Rfactor Rfree error: 0.019 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.384 412 10.4 %
Rwork0.343 3532 -
obs--94 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein_rep.param
X-RAY DIFFRACTION2water_rep.paramwater.top
X-RAY DIFFRACTION3hetero.hetero.
X-RAY DIFFRACTION4pat.parpat.top

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