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- PDB-7y9k: Crystal structure of P450 BM3-TMK from Bacillus megaterium -

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Basic information

Entry
Database: PDB / ID: 7y9k
TitleCrystal structure of P450 BM3-TMK from Bacillus megaterium
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / synthesis / monooxygenase
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 / FMN binding / iron ion binding / heme binding / identical protein binding / cytoplasm
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 / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.23 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: Chinese J Catal / Year: 2023
Title: Engineering of a P450-based Kemp eliminase with a new mechanism
Authors: Li, A. / Wang, Q. / Song, X. / Zhang, X. / Huang, J.W. / Chen, C.C. / Guo, R.T. / Wang, B. / Reetz, M.T.
History
DepositionJun 25, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 28, 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:
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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)107,2544
Polymers106,0212
Non-polymers1,2332
Water8,971498
1
A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,6272
Polymers53,0101
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1290 Å2
ΔGint-22 kcal/mol
Surface area19410 Å2
MethodPISA
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,6272
Polymers53,0101
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1290 Å2
ΔGint-21 kcal/mol
Surface area19390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.646, 147.961, 64.003
Angle α, β, γ (deg.)90.000, 98.480, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21(chain B and (resid 4 through 456 or resid 501))

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS / End auth comp-ID: HEM / End label comp-ID: HEM / Auth seq-ID: 4 - 501 / Label seq-ID: 4

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain AAA - C
2(chain B and (resid 4 through 456 or resid 501))BB - D

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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 / ...Cytochrome P450(BM-3) / Cytochrome P450BM-3 / Fatty acid monooxygenase / Flavocytochrome P450 BM3 / Cytochrome P450 BM3-TMK


Mass: 53010.391 Da / Num. of mol.: 2 / Mutation: E5K,L76Y,F88G,T439K
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 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase
#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 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 498 / 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: 2.63 Å3/Da / Density % sol: 53.19 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: 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
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.83→25 Å / Num. obs: 92441 / % possible obs: 98.6 % / Redundancy: 5.7 % / Rmerge(I) obs: 0.064 / Rpim(I) all: 0.031 / Rrim(I) all: 0.071 / Χ2: 1.286 / Net I/σ(I): 12 / Num. measured all: 524891
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.83-1.95.70.74693120.8850.3430.8230.63100
1.9-1.975.90.52593620.9390.2370.5770.71100
1.97-2.065.90.34893370.9690.1560.3820.794100
2.06-2.175.90.23593630.980.1060.2580.924100
2.17-2.315.90.16193390.9890.0730.1771.03499.9
2.31-2.485.80.11493210.9930.0520.1261.18399.7
2.48-2.735.90.08392500.9960.0380.0921.39198.8
2.73-3.135.60.05890850.9970.0270.0641.72496.6
3.13-3.945.10.04189490.9980.020.0462.22395.2
3.94-2550.03891230.9970.020.0432.74196.3

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Processing

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

4kpa


Resolution: 2.23→24.93 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 20.79 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2082 2000 3.83 %
Rwork0.1705 50244 -
obs0.1718 52244 99.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 95.56 Å2 / Biso mean: 36.6491 Å2 / Biso min: 20.16 Å2
Refinement stepCycle: final / Resolution: 2.23→24.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7312 0 86 498 7896
Biso mean--26.22 41.59 -
Num. residues----907
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2768X-RAY DIFFRACTION4.743TORSIONAL
12B2768X-RAY DIFFRACTION4.743TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Num. reflection Rfree: 200 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.23-2.310.26380.22664997519799
2.31-2.40.26820.212750285228100
2.4-2.510.24520.208650305230100
2.51-2.640.22980.198850185218100
2.64-2.810.2430.193350265226100
2.81-3.030.23040.198950335233100
3.03-3.330.25340.186450455245100
3.33-3.810.19880.162450515251100
3.81-4.790.16650.13954953515398
4.79-24.930.1710.14335063526399

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