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- PDB-6lvc: Structure of Dimethylformamidase, dimer -

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Basic information

Entry
Database: PDB / ID: 6lvc
TitleStructure of Dimethylformamidase, dimer
Components
  • N,N-dimethylformamidase large subunit
  • N,N-dimethylformamidase small subunit
KeywordsHYDROLASE / ab polypeptide / mononuclear iron / amidohydrolase / tetramer
Function / homology
Function and homology information


N,N-dimethylformamidase / N,N-dimethylformamidase activity / metal ion binding
Similarity search - Function
N,N-dimethylformamidase beta subunit-like, C-terminal / N,N-dimethylformamidase beta subunit-like, C-terminal / Concanavalin A-like lectin/glucanases superfamily / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
: / N,N-dimethylformamidase large subunit / N,N-dimethylformamidase small subunit
Similarity search - Component
Biological speciesParacoccus sp. SSG05 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsArya, C.A. / Yadav, S. / Fine, J. / Casanal, A. / Chopra, G. / Ramanathan, G. / Subramanian, R. / Vinothkumar, K.R.
Funding support India, 3items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Department of Biotechnology (DBT, India)BT/PR5081/INF/22/156/2012 India
Science and Engineering Research Board (SERB)SB/S2/RJN-094/2017 India
CitationJournal: Angew Chem Int Ed Engl / Year: 2020
Title: A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.
Authors: Chetan Kumar Arya / Swati Yadav / Jonathan Fine / Ana Casanal / Gaurav Chopra / Gurunath Ramanathan / Kutti R Vinothkumar / Ramaswamy Subramanian /
Abstract: N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved ...N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.
History
DepositionFeb 2, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 3, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 27, 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

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Assembly

Deposited unit
A: N,N-dimethylformamidase large subunit
B: N,N-dimethylformamidase small subunit
C: N,N-dimethylformamidase large subunit
D: N,N-dimethylformamidase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,9636
Polymers204,8514
Non-polymers1122
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17850 Å2
ΔGint-120 kcal/mol
Surface area55730 Å2

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Components

#1: Protein N,N-dimethylformamidase large subunit


Mass: 86341.758 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star / References: UniProt: I6NT79, N,N-dimethylformamidase
#2: Protein N,N-dimethylformamidase small subunit


Mass: 16083.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star / References: UniProt: I6NWZ0, N,N-dimethylformamidase
#3: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / 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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimethylformamidase / Type: COMPLEX / Details: Dimer, (a2b2) / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Paracoccus sp. SSG05 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3 (Star)
Buffer solutionpH: 7.2
Buffer componentConc.: 50 mM / Name: Tris-Cl
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample at no salt exists mostly as dimer. Buffer used had no salt.
Specimen supportDetails: Glow discharge was carried out with Quorum glocube at 25 mA for 90 seconds
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K
Details: Blot force was 0 and blotting was done for 3.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 5148 nm / Nominal defocus min: 1526 nm / Calibrated defocus min: 1526 nm / Calibrated defocus max: 5148 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77.7 K / Temperature (min): 77.7 K
Image recordingAverage exposure time: 60 sec. / Electron dose: 27 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1147
Details: A total of 25 frames were saved from the 60 second exposure, resulting in ~1.08 electron/frame
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
2EPU1.9image acquisition
4Gctf1.06CTF correctionGctf was used to estimate CTF values
7Coot0.9-premodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.13model refinement
CTF correctionDetails: CTF correction was done with Relion, during refinement and reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 169988
Details: Two rounds of 2D classification was performed prior to angular assignment and reconstruction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80674 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 42 / Protocol: OTHER / Space: REAL

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