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- PDB-9ist: Crystal Structure of Cytochrome P450BM3 VI-18A12 Mutant Heme Doma... -

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Basic information

Entry
Database: PDB / ID: 9ist
TitleCrystal Structure of Cytochrome P450BM3 VI-18A12 Mutant Heme Domain with N-Decanoyl-L-Homoserine Lactone
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / P450 / Monooxygenase / Decoy molecules
Function / homology
Function and homology information


aromatase activity / 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 / 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 / N-[(3S)-2-oxotetrahydrofuran-3-yl]decanamide / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.27 Å
AuthorsYokoyama, Y. / Sugimoto, H. / Shoji, O.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP22H05129 Japan
Japan Society for the Promotion of Science (JSPS)JP21H04704 Japan
CitationJournal: Chemcatchem / Year: 2024
Title: Bacterial Acyl Homoserine Lactones Triggered Non-Native Substrate Hydroxylation Catalyzed by Directed-Evolution-Derived Cytochrome P450BM3 Mutants
Authors: Yokoyama, Y. / Ariyasu, S. / Karasawa, M. / Kasai, C. / Aiba, Y. / Sugimoto, H. / Shoji, O.
History
DepositionJul 18, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)106,99415
Polymers104,4492
Non-polymers2,54513
Water6,449358
1
A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,3596
Polymers52,2251
Non-polymers1,1345
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1230 Å2
ΔGint-21 kcal/mol
Surface area19020 Å2
MethodPISA
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,6359
Polymers52,2251
Non-polymers1,4108
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1220 Å2
ΔGint-21 kcal/mol
Surface area19040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.482, 148.785, 61.374
Angle α, β, γ (deg.)90.00, 98.91, 90.00
Int Tables number4
Space group name H-MP1211

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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: 52224.555 Da / Num. of mol.: 2 / Mutation: P25V, R47W, T49L, A74V, L188P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Priestia megaterium (bacteria) / Gene: cyp102A1, cyp102, BG04_163 / Plasmid: pET28a(+) / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase

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Non-polymers , 5 types, 371 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-HL0 / N-[(3S)-2-oxotetrahydrofuran-3-yl]decanamide / N-decanoyl-L-homoserine lactone


Mass: 255.353 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H25NO3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 358 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: 2.6 Å3/Da / Density % sol: 52.69 %
Crystal growTemperature: 293 K / Method: batch mode / pH: 7.4
Details: PEG 8000, Tris-HCl, magnesium chloride, N-decanoyl homoserine lactone

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL45XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Jan 19, 2022
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.27→47 Å / Num. obs: 46574 / % possible obs: 97.6 % / Redundancy: 6.1 % / CC1/2: 0.97 / Rmerge(I) obs: 0.319 / Rpim(I) all: 0.141 / Rrim(I) all: 0.351 / Χ2: 1.03 / Net I/σ(I): 5.5 / Num. measured all: 282581
Reflection shellResolution: 2.27→2.34 Å / Redundancy: 6.2 % / Rmerge(I) obs: 1.327 / Num. unique obs: 4291 / CC1/2: 0.539 / Rpim(I) all: 0.582 / Rrim(I) all: 1.453 / Χ2: 0.99 / % possible all: 98

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
PDB_EXTRACTdata extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.27→47 Å / Cor.coef. Fo:Fc: 0.942 / Cor.coef. Fo:Fc free: 0.888 / SU B: 9.496 / SU ML: 0.218 / Cross valid method: THROUGHOUT / ESU R: 0.346 / ESU R Free: 0.255 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25946 2326 5 %RANDOM
Rwork0.18883 ---
obs0.19227 44238 97.46 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 24.172 Å2
Baniso -1Baniso -2Baniso -3
1--2.01 Å20 Å2-0.79 Å2
2---0.45 Å20 Å2
3---2.58 Å2
Refinement stepCycle: 1 / Resolution: 2.27→47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7352 0 172 358 7882
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0137703
X-RAY DIFFRACTIONr_bond_other_d0.0010.0167330
X-RAY DIFFRACTIONr_angle_refined_deg1.6981.66710430
X-RAY DIFFRACTIONr_angle_other_deg1.2751.59316936
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9935908
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.82523.463410
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.765151356
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.131538
X-RAY DIFFRACTIONr_chiral_restr0.0760.2952
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.028626
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021722
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.8162.4163638
X-RAY DIFFRACTIONr_mcbond_other1.8142.4153637
X-RAY DIFFRACTIONr_mcangle_it2.8113.6174544
X-RAY DIFFRACTIONr_mcangle_other2.8113.6194545
X-RAY DIFFRACTIONr_scbond_it2.2572.6514065
X-RAY DIFFRACTIONr_scbond_other2.2562.6514066
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.623.8755888
X-RAY DIFFRACTIONr_long_range_B_refined4.82627.958671
X-RAY DIFFRACTIONr_long_range_B_other4.80427.9298638
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.27→2.329 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.36 196 -
Rwork0.288 3263 -
obs--98.1 %

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