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- PDB-7y9l: Crystal structure of P450 BM3-2F from Bacillus megaterium in comp... -

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Basic information

Entry
Database: PDB / ID: 7y9l
TitleCrystal structure of P450 BM3-2F from Bacillus megaterium in complex with 2-Hydroxy-5-Nitrobenzonitrile
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / synthesis / monooxygenase
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 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / 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 / Flavoprotein-like superfamily / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450
Similarity search - Domain/homology
5-nitro-2-oxidanyl-benzenecarbonitrile / PROTOPORPHYRIN IX CONTAINING FE / NICKEL (II) ION / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.76 Å
AuthorsWang, Q. / Zhang, L.L. / Liu, W.D. / Huang, J.-W. / Yang, Y. / Chen, C.-C. / Guo, R.-T.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: to be published
Title: Crystal structure of P450 BM3-2F from Bacillus megaterium in complex with 2-Hydroxy-5-Nitrobenzonitrile
Authors: Wang, Q. / Zhang, L.L. / Liu, W.D. / Huang, J.-W. / Yang, Y. / Chen, C.-C. / Guo, R.-T.
History
DepositionJun 25, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 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 cytochrome P450/NADPH--P450 reductase
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,4908
Polymers104,8112
Non-polymers1,6796
Water12,322684
1
A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2454
Polymers52,4061
Non-polymers8393
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1270 Å2
ΔGint-21 kcal/mol
Surface area19110 Å2
MethodPISA
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2454
Polymers52,4061
Non-polymers8393
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1280 Å2
ΔGint-21 kcal/mol
Surface area18970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.014, 146.956, 63.271
Angle α, β, γ (deg.)90.000, 98.070, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 5 through 52 or resid 54...
21(chain B and (resid 5 through 40 or (resid 41...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11METMETLEULEU(chain A and (resid 5 through 52 or resid 54...AA5 - 525 - 52
12SERSERLEULEU(chain A and (resid 5 through 52 or resid 54...AA54 - 26254 - 262
13ALAALATHRTHR(chain A and (resid 5 through 52 or resid 54...AA264 - 269264 - 269
14GLYGLYGLNGLN(chain A and (resid 5 through 52 or resid 54...AA271 - 403271 - 403
15PHEPHELEULEU(chain A and (resid 5 through 52 or resid 54...AA405 - 455405 - 455
166VP6VP6VP6VP(chain A and (resid 5 through 52 or resid 54...AC1001
21METMETPHEPHE(chain B and (resid 5 through 40 or (resid 41...BB5 - 405 - 40
22LYSLYSLYSLYS(chain B and (resid 5 through 40 or (resid 41...BB4141
23METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
24METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
25METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
26METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
27METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
28METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455
29METMETLEULEU(chain B and (resid 5 through 40 or (resid 41...BB5 - 4555 - 455

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Components

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


Mass: 52405.730 Da / Num. of mol.: 2 / Mutation: A82F,A328F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Priestia megaterium (bacteria)
Strain: ATCC 14581 / DSM 32 / CCUG 1817 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / NRRL B-14308 / VKM B-512 / Ford 19
Gene: cyp102A1, cyp102, BG04_163 / Plasmid: pRSF-DuetI / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase
#2: Chemical ChemComp-6VP / 5-nitro-2-oxidanyl-benzenecarbonitrile / 2-hydroxy-5-nitrobenzonitrile


Mass: 164.118 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H4N2O3 / Feature type: SUBJECT OF INVESTIGATION
#3: 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
#4: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 684 / 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: 2.59 Å3/Da / Density % sol: 52.54 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: 25-30% PEG 3350, 0.1 M MgCl2, 0.1 M HEPES, pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: BL13B1 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 8, 2019
RadiationMonochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.76→25 Å / Num. obs: 100707 / % possible obs: 95.7 % / Redundancy: 5.3 % / Rmerge(I) obs: 0.049 / Rpim(I) all: 0.023 / Rrim(I) all: 0.055 / Χ2: 1.981 / Net I/σ(I): 19.3 / Num. measured all: 530937
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.76-1.824.80.42993460.8570.2140.4810.90289.3
1.82-1.95.50.30395420.9420.1390.3341.0391.1
1.9-1.985.50.21498120.9730.0960.2351.18193.1
1.98-2.095.40.14299930.9860.0650.1561.41295.2
2.09-2.225.40.104101240.9920.0480.1151.67396.4
2.22-2.395.30.083102230.9950.0390.0922.10497.3
2.39-2.635.30.067103060.9960.0320.0742.48797.9
2.63-3.015.20.053103880.9970.0250.0592.79598.5
3.01-3.795.10.041104720.9980.020.0452.97999.3
3.79-255.20.038105010.9980.0180.0423.04498.6

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-2000data scaling
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4KPA

4kpa


Resolution: 1.76→24.78 Å / SU ML: 0.17 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 20.55 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2031 2012 2 %
Rwork0.173 98598 -
obs0.1736 100610 95.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 111.19 Å2 / Biso mean: 36.5465 Å2 / Biso min: 17.14 Å2
Refinement stepCycle: final / Resolution: 1.76→24.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7250 0 112 684 8046
Biso mean--26.29 42.83 -
Num. residues----896
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2694X-RAY DIFFRACTION6.016TORSIONAL
12B2694X-RAY DIFFRACTION6.016TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.76-1.820.25272010.239073927489
1.82-1.90.22241890.20219345953491
1.9-1.980.22781770.19789639981693
1.98-2.090.24292130.188397881000195
2.09-2.220.23972030.182999101011396
2.22-2.390.21111960.1784100361023297
2.39-2.630.22322030.181101041030798
2.63-3.010.21362110.1855101721038399
3.01-3.790.19082110.164102601047199
3.79-24.780.17822080.1562102711047998

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