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- PDB-6lvv: N, N-dimethylformamidase -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 6lvv
TitleN, N-dimethylformamidase
Components
  • N,N-dimethylformamidase large subunit
  • N,N-dimethylformamidase small subunit
KeywordsHYDROLASE / Amidase / Linear aliphatic amidohydrolase / metallo-amidase / heterotetramer.
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsArya, C.K. / Ramaswamy, S. / Kutti, R.V. / Gurunath, R.
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 5, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 24, 2021Group: Database references / Category: citation / citation_author
Revision 1.3Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
I: N,N-dimethylformamidase large subunit
J: N,N-dimethylformamidase small subunit
K: N,N-dimethylformamidase large subunit
L: N,N-dimethylformamidase small subunit
M: N,N-dimethylformamidase large subunit
N: N,N-dimethylformamidase small subunit
O: N,N-dimethylformamidase large subunit
P: N,N-dimethylformamidase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)820,34832
Polymers819,40516
Non-polymers94316
Water3,153175
1
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)205,0878
Polymers204,8514
Non-polymers2364
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18640 Å2
ΔGint-115 kcal/mol
Surface area53050 Å2
MethodPISA
2
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)205,0878
Polymers204,8514
Non-polymers2364
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18650 Å2
ΔGint-115 kcal/mol
Surface area53040 Å2
MethodPISA
3
I: N,N-dimethylformamidase large subunit
J: N,N-dimethylformamidase small subunit
K: N,N-dimethylformamidase large subunit
L: N,N-dimethylformamidase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,0878
Polymers204,8514
Non-polymers2364
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18570 Å2
ΔGint-115 kcal/mol
Surface area53110 Å2
MethodPISA
4
M: N,N-dimethylformamidase large subunit
N: N,N-dimethylformamidase small subunit
O: N,N-dimethylformamidase large subunit
P: N,N-dimethylformamidase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,0878
Polymers204,8514
Non-polymers2364
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18670 Å2
ΔGint-118 kcal/mol
Surface area53030 Å2
MethodPISA
Unit cell
Length a, b, c (Å)160.514, 142.553, 181.488
Angle α, β, γ (deg.)90.000, 92.920, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A or chain B
21chain G or chain H
31chain I or chain J
41chain O or chain P
12chain C or chain D
22chain E or chain F
32chain K or chain L
42chain M or chain N

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111METMETVALVALchain A or chain BAA1 - 7621 - 762
121SERSERLEULEUchain A or chain BBB7 - 1317 - 131
211METMETVALVALchain G or chain HGG1 - 7621 - 762
221SERSERLEULEUchain G or chain HHH7 - 1317 - 131
311METMETVALVALchain I or chain JII1 - 7621 - 762
321SERSERLEULEUchain I or chain JJJ7 - 1317 - 131
411METMETVALVALchain O or chain POO1 - 7621 - 762
421SERSERLEULEUchain O or chain PPP7 - 1317 - 131
112METMETVALVALchain C or chain DCC1 - 7621 - 762
122SERSERLEULEUchain C or chain DDD7 - 1317 - 131
212METMETVALVALchain E or chain FEE1 - 7621 - 762
222SERSERLEULEUchain E or chain FFF7 - 1317 - 131
312METMETVALVALchain K or chain LKK1 - 7621 - 762
322SERSERLEULEUchain K or chain LLL7 - 1317 - 131
412METMETVALVALchain M or chain NMM1 - 7621 - 762
422SERSERLEULEUchain M or chain NNN7 - 1317 - 131

NCS ensembles :
ID
1
2

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Components

#1: Protein
N,N-dimethylformamidase large subunit


Mass: 86341.758 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase2 / Plasmid: pET21a(+) / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / References: UniProt: I6NT79, N,N-dimethylformamidase
#2: Protein
N,N-dimethylformamidase small subunit


Mass: 16083.823 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase1 / Plasmid: pET21a(+) / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / References: UniProt: I6NWZ0, N,N-dimethylformamidase
#3: Chemical
ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 175 / 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: 2.53 Å3/Da / Density % sol: 51.41 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.2
Details: PEG 3350, 16%; Tris-HCl buffer pH 7.2, 50 mM, NaCl 200 mM; KCl 200 mM, Protein 5 mg/ml.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.0053 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Mar 10, 2018
RadiationMonochromator: Si(III) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0053 Å / Relative weight: 1
ReflectionResolution: 2.8→49.47 Å / Num. obs: 200660 / % possible obs: 99.75 % / Redundancy: 2 % / Biso Wilson estimate: 26.91 Å2 / CC1/2: 0.93 / CC star: 0.982 / Net I/σ(I): 4.44
Reflection shellResolution: 2.8→2.87 Å / Num. unique obs: 9942 / CC1/2: 0.321 / Rpim(I) all: 0.789 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX1.18rc6_3830refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6LVC

6lvc


Resolution: 2.8→49.47 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 27.13 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2666 1920 -Random
Rwork0.2265 198321 --
obs0.2268 200241 99.75 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 133.58 Å2 / Biso mean: 30.2851 Å2 / Biso min: 5.91 Å2
Refinement stepCycle: final / Resolution: 2.8→49.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms56576 0 88 175 56839
Biso mean--26.5 15.7 -
Num. residues----7096
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A7077X-RAY DIFFRACTIONPOSITIONAL0.078
12G7077X-RAY DIFFRACTIONPOSITIONAL0.078
13I7077X-RAY DIFFRACTIONPOSITIONAL0.076
14O7077X-RAY DIFFRACTIONPOSITIONAL0.056
21C7077X-RAY DIFFRACTIONPOSITIONAL0.11
22E7077X-RAY DIFFRACTIONPOSITIONAL0.11
23K7077X-RAY DIFFRACTIONPOSITIONAL0.07
24M7077X-RAY DIFFRACTIONPOSITIONAL0.036
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.8-2.870.3541570.31741411114268100
2.87-2.950.33871590.29731407714236100
2.95-3.030.32061320.28891420814340100
3.03-3.130.33681470.28261409014237100
3.13-3.240.3131440.27611416114305100
3.24-3.370.29851640.24361408614250100
3.37-3.530.31911260.26661411714243100
3.53-3.710.31271430.2848140361417999
3.71-3.950.31461290.2638140181414799
3.95-4.250.19191150.18551420514320100
4.25-4.680.17131400.15491420914349100
4.68-5.350.19461220.15071425814380100
5.35-6.740.19421260.17081429514421100
6.74-49.470.17821160.15161445014566100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.05750.21510.06710.64580.17780.3750.04120.12110.0355-0.012-0.0779-0.073-0.0105-0.0221-0.00250.1420.0361-0.01120.10080.01870.176693.64677.852221.6303
20.25830.05910.0890.2379-0.01010.3968-0.06380.13050.0324-0.2375-0.07870.0625-0.2527-0.0305-0.08220.35090.0582-0.02590.21410.09710.35584.9462105.075813.5274
30.8019-0.27250.27340.5582-0.13720.4853-0.0322-0.18760.16120.0613-0.03870.0241-0.1523-0.1377-0.01260.23620.0092-0.02090.1956-0.07920.193979.657791.602957.591
40.07130.0138-0.1340.4426-0.18710.4330.0275-0.05660.3152-0.0389-0.1517-0.1231-0.35290.1436-0.22550.45030.072-0.12280.0062-0.1540.594193.6799115.932747.9676
50.90470.3526-0.02210.6555-0.19640.3913-0.01940.0245-0.1703-0.0208-0.0047-0.11590.0515-0.0289-0.00780.12930.02-0.01810.1039-0.00260.216781.393830.572733.0958
60.20390.02670.13220.0894-0.00850.39670.06980.0089-0.24570.0495-0.0698-0.27450.20140.13550.12380.2630.00860.04820.03890.20680.775294.66226.435544.1604
70.72360.2762-0.21190.7132-0.33690.50220.1273-0.3935-0.15330.2601-0.2357-0.1647-0.0294-0.005-0.0890.1692-0.1085-0.2020.28520.11090.164691.135344.548470.2579
80.25630.1650.12630.42030.19730.5788-0.167-0.2032-0.06080.2302-0.1222-0.05170.0572-0.0445-0.66150.3816-0.175-0.07550.34530.36340.353181.984117.223877.4793
90.71320.1659-0.05250.43080.08510.39480.0070.07570.0301-0.12370.00410.0133-0.0454-0.041400.17880.03390.0170.18140.0210.148214.091241.549522.1632
100.19760.02190.03770.3337-0.05680.27510.04370.02130.0835-0.0998-0.04020.0518-0.1749-0.1083-0.04750.34880.06870.06650.25440.05650.19666.311369.250614.7661
110.4704-0.310.04950.3669-0.18780.38480.0178-0.08390.07020.10380.0491-0.0364-0.1164-0.12720.02140.210.06220.0650.1781-0.01660.12510.787954.925758.4908
120.31360.0022-0.00270.3404-0.11770.18570.0287-0.08530.26840.04310.0218-0.2691-0.15380.1197-0.00640.42460.02310.0680.2408-0.07510.265715.56479.062349.5422
130.63650.2814-0.05820.4975-0.06250.37140.01490.006-0.0545-0.0368-0.00020.00830.1347-0.09380.00010.2483-0.0307-0.00910.16930.0070.10610.2955-5.489132.4637
140.2867-0.14150.02010.3204-0.03970.19290.01170.0232-0.1587-0.0305-0.0489-0.08540.30510.0413-0.00860.4599-0.03180.02060.1456-0.00420.247612.7207-30.318242.9621
150.6547-0.04020.03060.4438-0.10320.4175-0.0347-0.0495-0.02940.09960.02750.00520.0649-0.07850.00020.187-0.02820.01230.19640.00570.126610.7667.353569.8716
160.23990.03960.00990.2704-0.07180.66510.043-0.1513-0.17630.09990.00870.12420.2401-0.3653-0.11380.4245-0.1774-0.00280.22860.1190.21030.6891-19.794976.5007
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 761)A1 - 761
2X-RAY DIFFRACTION2chain 'B' and (resid 7 through 131)B7 - 131
3X-RAY DIFFRACTION3chain 'C' and (resid 1 through 761 )C1 - 761
4X-RAY DIFFRACTION4chain 'D' and (resid 7 through 131 )D7 - 131
5X-RAY DIFFRACTION5chain 'E' and (resid 1 through 761)E1 - 761
6X-RAY DIFFRACTION6chain 'F' and (resid 7 through 131)F7 - 131
7X-RAY DIFFRACTION7chain 'G' and (resid 1 through 761 )G1 - 761
8X-RAY DIFFRACTION8chain 'H' and (resid 7 through 131)H7 - 131
9X-RAY DIFFRACTION9chain 'I' and (resid 1 through 761 )I1 - 761
10X-RAY DIFFRACTION10chain 'J' and (resid 7 through 131 )J7 - 131
11X-RAY DIFFRACTION11chain 'K' and (resid 1 through 761 )K1 - 761
12X-RAY DIFFRACTION12chain 'L' and (resid 7 through 131 )L7 - 131
13X-RAY DIFFRACTION13chain 'M' and (resid 1 through 761 )M1 - 761
14X-RAY DIFFRACTION14chain 'N' and (resid 7 through 131)N7 - 131
15X-RAY DIFFRACTION15chain 'O' and (resid 1 through 761 )O1 - 761
16X-RAY DIFFRACTION16chain 'P' and (resid 7 through 131 )P7 - 131

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