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- PDB-2fm7: Evolution of Enzymatic Activity in the Tautomerase Superfamily: M... -

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Basic information

Entry
Database: PDB / ID: 2fm7
TitleEvolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase
Components4-Oxalocrotonate Tautomerase
KeywordsTRANSFERASE / 4-Oxalocrotonate / tautomerase / 4-OT / homo-hexamer / dehalogenase / mutant / L8R
Function / homology
Function and homology information


xylene catabolic process / 2-hydroxymuconate tautomerase / toluene catabolic process / isomerase activity
Similarity search - Function
4-oxalocrotonate tautomerase, Pseudomonas-type / 4-oxalocrotonate tautomerase / Tautomerase enzyme / Macrophage Migration Inhibitory Factor / Macrophage Migration Inhibitory Factor / Tautomerase/MIF superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
2-hydroxymuconate tautomerase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsAlmrud, J.J. / Hackert, M.L.
CitationJournal: Biochemistry / Year: 2006
Title: Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase
Authors: Poelarends, G.J. / Almrud, J.J. / Serrano, H. / Darty, J.E. / Johnson, W.H. / Hackert, M.L. / Whitman, C.P.
History
DepositionJan 8, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 26, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.end_auth_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 4-Oxalocrotonate Tautomerase
B: 4-Oxalocrotonate Tautomerase
C: 4-Oxalocrotonate Tautomerase
D: 4-Oxalocrotonate Tautomerase
E: 4-Oxalocrotonate Tautomerase
F: 4-Oxalocrotonate Tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,6307
Polymers40,5946
Non-polymers351
Water3,585199
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: 4-Oxalocrotonate Tautomerase
B: 4-Oxalocrotonate Tautomerase

A: 4-Oxalocrotonate Tautomerase
B: 4-Oxalocrotonate Tautomerase

A: 4-Oxalocrotonate Tautomerase
B: 4-Oxalocrotonate Tautomerase


Theoretical massNumber of molelcules
Total (without water)40,5946
Polymers40,5946
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area12970 Å2
ΔGint-65 kcal/mol
Surface area15230 Å2
MethodPISA
3
E: 4-Oxalocrotonate Tautomerase
F: 4-Oxalocrotonate Tautomerase

E: 4-Oxalocrotonate Tautomerase
F: 4-Oxalocrotonate Tautomerase

E: 4-Oxalocrotonate Tautomerase
F: 4-Oxalocrotonate Tautomerase


Theoretical massNumber of molelcules
Total (without water)40,5946
Polymers40,5946
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area12880 Å2
ΔGint-68 kcal/mol
Surface area14590 Å2
MethodPISA
4
A: 4-Oxalocrotonate Tautomerase
B: 4-Oxalocrotonate Tautomerase


Theoretical massNumber of molelcules
Total (without water)13,5312
Polymers13,5312
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
5
E: 4-Oxalocrotonate Tautomerase
F: 4-Oxalocrotonate Tautomerase


Theoretical massNumber of molelcules
Total (without water)13,5312
Polymers13,5312
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
6
C: 4-Oxalocrotonate Tautomerase
D: 4-Oxalocrotonate Tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,5673
Polymers13,5312
Non-polymers351
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)80.863, 80.863, 117.038
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number173
Space group name H-MP63
Components on special symmetry positions
IDModelComponents
11A-87-

HOH

21A-88-

HOH

31B-94-

HOH

41B-96-

HOH

51C-5010-

HOH

61D-9020-

HOH

71E-71-

HOH

81E-185-

HOH

91F-84-

HOH

101F-85-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D
51E
61F

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / Refine code: 1

Dom-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1GLYGLYAA1 - 621 - 62
2GLYGLYBB1 - 621 - 62
3ARGARGCC1 - 611 - 61
4ARGARGDD1 - 611 - 61
5ARGARGEE1 - 611 - 61
6ARGARGFF1 - 611 - 61
DetailsThe homo-hexamer biological assembly is generated from application of the space group's crystallographic symmetry operators to the dimers in the asymmetric.

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Components

#1: Protein
4-Oxalocrotonate Tautomerase / E.C.5.3.2.- / 4-OT


Mass: 6765.732 Da / Num. of mol.: 6 / Mutation: L8R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Strain: mt-2 / Description: used TOL plasmid pWW0 / Gene: xylH / Plasmid: pET3b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21Gold(DE3)
References: UniProt: Q01468, Isomerases; Intramolecular oxidoreductases; Interconverting keto- and enol-groups
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 199 / 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: 2.72 Å3/Da / Density % sol: 54.78 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 3 microlitres of protein (20 mg/mL solution in 10 mM Tris-Cl, pH 7.0) mixed with an equal volume of reservoir buffer [30% O-(2-aminopropyl)-O-(2-methoxyethyl)polypropylene glycol 500, 100 mM ...Details: 3 microlitres of protein (20 mg/mL solution in 10 mM Tris-Cl, pH 7.0) mixed with an equal volume of reservoir buffer [30% O-(2-aminopropyl)-O-(2-methoxyethyl)polypropylene glycol 500, 100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, and 50 mM CsCl]. The resulting mixture was allowed to equilibrate against 50 microlitres of reservoir solution, VAPOR DIFFUSION, SITTING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU300 / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS HTC / Detector: IMAGE PLATE / Date: Feb 27, 2004
RadiationMonochromator: Osmic confocal optics / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.8→50 Å / Num. all: 10782 / Num. obs: 10209 / % possible obs: 99.9 % / Observed criterion σ(I): 2 / Redundancy: 7.8 % / Biso Wilson estimate: 35.1 Å2 / Rmerge(I) obs: 0.12 / Rsym value: 0.162 / Net I/σ(I): 5.7
Reflection shellResolution: 2.8→2.9 Å / Redundancy: 7.7 % / Rmerge(I) obs: 0.401 / Rsym value: 0.424 / % possible all: 100

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Processing

Software
NameVersionClassification
HKL-2000data collection
SCALEPACKdata scaling
MOLREPphasing
REFMAC5.1.24refinement
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1BJP with coordinates for OXP removed

1bjp


Resolution: 2.8→34 Å / Cor.coef. Fo:Fc: 0.903 / Cor.coef. Fo:Fc free: 0.799 / SU B: 21.345 / SU ML: 0.399 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R Free: 0.463 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.30109 514 4.8 %RANDOM
Rwork0.22979 ---
all0.23327 10726 --
obs0.23327 10209 99.97 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 25.763 Å2
Baniso -1Baniso -2Baniso -3
1-1.3 Å20.65 Å20 Å2
2--1.3 Å20 Å2
3----1.95 Å2
Refinement stepCycle: LAST / Resolution: 2.8→34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2769 0 1 199 2969
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0240.0212808
X-RAY DIFFRACTIONr_angle_refined_deg2.3571.9633770
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9835366
X-RAY DIFFRACTIONr_chiral_restr0.1150.2449
X-RAY DIFFRACTIONr_gen_planes_refined0.0170.022029
X-RAY DIFFRACTIONr_nbd_refined0.2480.21162
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.3250.2188
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2910.2146
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.0960.225
X-RAY DIFFRACTIONr_mcbond_it1.2571.51827
X-RAY DIFFRACTIONr_mcangle_it2.33822924
X-RAY DIFFRACTIONr_scbond_it3.1863981
X-RAY DIFFRACTIONr_scangle_it5.8854.5846
Refine LS restraints NCS

Ens-ID: 1 / Number: 363 / Refine-ID: X-RAY DIFFRACTION

Dom-IDAuth asym-IDTypeRms dev position (Å)Weight position
1Atight positional0.140.05
2Btight positional0.180.05
3Ctight positional0.260.05
4Dtight positional0.210.05
5Etight positional0.220.05
6Ftight positional0.150.05
1Atight thermal0.130.5
2Btight thermal0.130.5
3Ctight thermal0.110.5
4Dtight thermal0.070.5
5Etight thermal0.110.5
6Ftight thermal0.080.5
LS refinement shellResolution: 2.8→2.872 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.326 36 -
Rwork0.271 736 -
obs-736 100 %

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