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- PDB-6lvb: Structure of Dimethylformamidase, tetramer -

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Basic information

Entry
Database: PDB / ID: 6lvb
TitleStructure of Dimethylformamidase, tetramer
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: 2.8 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
Citation
Journal: 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.
#1: Journal: Prog Biophys Mol Biol / Year: 2021
Title: Comparison of CryoEM and X-ray structures of dimethylformamidase.
Authors: Kutti R Vinothkumar / Chetan Kumar Arya / Gurunath Ramanathan / Ramaswamy Subramanian /
Abstract: Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of ...Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 °C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.
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 24, 2021Group: Database references / Category: citation / citation_author
Revision 1.4Mar 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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  • Deposited structure unit
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Structure viewerMolecule:
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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
E: N,N-dimethylformamidase large subunit
F: N,N-dimethylformamidase small subunit
G: N,N-dimethylformamidase large subunit
H: N,N-dimethylformamidase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)409,92612
Polymers409,7028
Non-polymers2234
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, At low concentration, the molecules can disassemble to form dimer containing 2x alpha+beta
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38630 Å2
ΔGint-260 kcal/mol
Surface area108560 Å2

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Components

#1: Protein
N,N-dimethylformamidase large subunit


Mass: 86341.758 Da / Num. of mol.: 4
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.: 4
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.: 4 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: Tetramer, 2x(a2b2) / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.4 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 component
IDConc.NameBuffer-ID
150 mMTris-Cl1
2200 mMNaCl1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample at higher salt exists mostly as tetramer
Specimen supportDetails: Glow discharge was carried out in Quorum Glocube instrument at 25mA 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: 4145.7 nm / Nominal defocus min: 931.5 nm / Calibrated defocus min: 931.5 nm / Calibrated defocus max: 4145.7 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: 793
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: 89183
Details: Two rounds of 2D classification was performed prior to angular assignment and reconstruction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59191 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 38.8 / Protocol: OTHER / Space: REAL

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