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- PDB-6bz5: Structure and mechanism of salicylate hydroxylase from Pseudomona... -

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Basic information

Entry
Database: PDB / ID: 6bz5
TitleStructure and mechanism of salicylate hydroxylase from Pseudomonas putida G7
ComponentsSalicylate hydroxylase
KeywordsOXIDOREDUCTASE / Aromatic hydrocarbons catabolism / salicylate hydroxylase / flavoprotein
Function / homology
Function and homology information


salicylate 1-monooxygenase / salicylate 1-monooxygenase activity / : / FAD binding
Similarity search - Function
Salicylate 1-monooxygenase / FAD-binding domain / FAD binding domain / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
IODIDE ION / Salicylate hydroxylase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.006 Å
AuthorsNagem, R.A.P. / Costa, D.M.A.
Funding support Brazil, 3items
OrganizationGrant numberCountry
FAPEMIGEDT-0185-0.00-07, Rede-170/08, APQ-01006-13 and RED-00010-14 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)INCT-Catalysis, 482173/2010-6, 306498/2013-8 and 484232/2013-4 Brazil
VALE S.A.RDP-00174-10 Brazil
CitationJournal: Int.J.Biol.Macromol. / Year: 2019
Title: Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.
Authors: Costa, D.M.A. / Gomez, S.V. / de Araujo, S.S. / Pereira, M.S. / Alves, R.B. / Favaro, D.C. / Hengge, A.C. / Nagem, R.A.P. / Brandao, T.A.S.
History
DepositionDec 22, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 26, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 8, 2020Group: Author supporting evidence / Database references / Category: citation / citation_author / pdbx_audit_support
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Salicylate hydroxylase
B: Salicylate hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,36038
Polymers98,0402
Non-polymers3,32136
Water8,323462
1
A: Salicylate hydroxylase
hetero molecules


  • defined by author&software
  • Evidence: gel filtration, The 6xHis-NahG exists as a monomer in solution according to size exclusion chromatography and dynamic light scattering assays, which is in agreement with literature ...Evidence: gel filtration, The 6xHis-NahG exists as a monomer in solution according to size exclusion chromatography and dynamic light scattering assays, which is in agreement with literature observations for the native enzyme., light scattering, The 6xHis-NahG exists as a monomer in solution according to size exclusion chromatography and dynamic light scattering assays, which is in agreement with literature observations for the native enzyme.
  • 51.2 kDa, 1 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)51,20825
Polymers49,0201
Non-polymers2,18824
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Salicylate hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,15313
Polymers49,0201
Non-polymers1,13312
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)81.615, 98.076, 130.136
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Salicylate hydroxylase / Salicylate 1-monooxygenase


Mass: 49019.879 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: nahG / Plasmid: pET28a(TEV)
Details (production host): pET28a(TEV) is a modified vector from pET28a (Novagen). The thrombin recognition site of pET28a (Novagen) was replaced by a TEV protease recognition site.
Production host: Escherichia coli (E. coli) / Strain (production host): RosettaTM (Novagen) / References: UniProt: P23262, salicylate 1-monooxygenase

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Non-polymers , 5 types, 498 molecules

#2: Chemical
ChemComp-IOD / IODIDE ION


Mass: 126.904 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: I
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 462 / 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: 1

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Sample preparation

CrystalDensity Matthews: 2.7 Å3/Da / Density % sol: 54.5 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 9
Details: Crystal was obtained manually by the hanging-drop vapour-diffusion after optimization of the initial conditions. The optimum condition was 1.26 M ammonium sulfate, 0.1 M Tris pH 9.0, 20% v/v glycerol.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: LNLS / Beamline: W01B-MX2 / Wavelength: 1.459 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Jul 5, 2012
Details: Cylindrical Vertical Collimating Mirror & Toroidal Horizontal and Vertical Focusing Mirror
RadiationMonochromator: Double Crystal Monochromator of Si-111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.459 Å / Relative weight: 1
ReflectionResolution: 2→40.81 Å / Num. obs: 133525 / % possible obs: 99 % / Redundancy: 7.1 % / Rmerge(I) obs: 0.064 / Net I/σ(I): 25.1
Reflection shellResolution: 2→2.03 Å / Redundancy: 5.2 % / Rmerge(I) obs: 0.526 / Mean I/σ(I) obs: 2.9 / Num. unique obs: 6476 / % possible all: 95.6

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Processing

Software
NameVersionClassification
PHENIX1.7.3-928refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIX1.7.3-928phasing
RefinementMethod to determine structure: SAD / Resolution: 2.006→40.81 Å / SU ML: 0.22 / Cross valid method: FREE R-VALUE / σ(F): 1.91 / Phase error: 25.44
RfactorNum. reflection% reflectionSelection details
Rfree0.2336 6750 5.09 %RANDOM SELECTION
Rwork0.2109 ---
obs0.212 132697 98.17 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.006→40.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6397 0 126 462 6985
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0026692
X-RAY DIFFRACTIONf_angle_d0.4719077
X-RAY DIFFRACTIONf_dihedral_angle_d2.6134563
X-RAY DIFFRACTIONf_chiral_restr0.04968
X-RAY DIFFRACTIONf_plane_restr0.0031201
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.0057-2.02850.32622220.30783726X-RAY DIFFRACTION88
2.0285-2.05240.32262080.30784118X-RAY DIFFRACTION97
2.0524-2.07740.28792260.2614188X-RAY DIFFRACTION98
2.0774-2.10370.28952050.26734228X-RAY DIFFRACTION99
2.1037-2.13140.27281880.26344257X-RAY DIFFRACTION98
2.1314-2.16060.25652110.2694217X-RAY DIFFRACTION99
2.1606-2.19150.26572180.26674195X-RAY DIFFRACTION99
2.1915-2.22420.29112580.26714193X-RAY DIFFRACTION98
2.2242-2.25890.30522080.26534227X-RAY DIFFRACTION99
2.2589-2.29590.31122530.25924155X-RAY DIFFRACTION98
2.2959-2.33550.34492360.25984236X-RAY DIFFRACTION98
2.3355-2.3780.29372300.25634200X-RAY DIFFRACTION99
2.378-2.42370.27192560.24284170X-RAY DIFFRACTION98
2.4237-2.47320.23842160.24514265X-RAY DIFFRACTION99
2.4732-2.5270.27972100.24794173X-RAY DIFFRACTION98
2.527-2.58570.28472630.2524203X-RAY DIFFRACTION99
2.5857-2.65040.28852560.2454212X-RAY DIFFRACTION98
2.6504-2.7220.28451910.23624229X-RAY DIFFRACTION98
2.722-2.80210.28262260.22954224X-RAY DIFFRACTION98
2.8021-2.89250.32032250.24224203X-RAY DIFFRACTION98
2.8925-2.99590.2452310.22864212X-RAY DIFFRACTION99
2.9959-3.11580.24871770.2234281X-RAY DIFFRACTION99
3.1158-3.25750.2392430.21474216X-RAY DIFFRACTION99
3.2575-3.42920.22492250.21634208X-RAY DIFFRACTION99
3.4292-3.64390.22742540.19454216X-RAY DIFFRACTION99
3.6439-3.92510.16982110.18294276X-RAY DIFFRACTION100
3.9251-4.31970.20382350.17044244X-RAY DIFFRACTION100
4.3197-4.94380.16912320.16394273X-RAY DIFFRACTION100
4.9438-6.22510.19632250.18644265X-RAY DIFFRACTION100
6.2251-40.81590.22922110.19744137X-RAY DIFFRACTION96

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