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- PDB-5clo: Crystal structure of a 4-oxalocrotonate tautomerase mutant in com... -

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Basic information

Entry
Database: PDB / ID: 5clo
TitleCrystal structure of a 4-oxalocrotonate tautomerase mutant in complex with nitrostyrene at 2.3 Angstrom
Components2-hydroxymuconate tautomerase
KeywordsISOMERASE / 4-Oxalocrotonate tautomerase / beta-alpha-beta structural motif / tautomerase superfamily
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
trans beta nitrostyrene / 2-hydroxymuconate tautomerase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.3 Å
AuthorsThunnissen, A.M.W.H. / Poddar, H.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
European Research Council242293 Netherlands
CitationJournal: Nat Commun / Year: 2016
Title: Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases.
Authors: van der Meer, J.Y. / Poddar, H. / Baas, B.J. / Miao, Y. / Rahimi, M. / Kunzendorf, A. / van Merkerk, R. / Tepper, P.G. / Geertsema, E.M. / Thunnissen, A.M. / Quax, W.J. / Poelarends, G.J.
History
DepositionJul 16, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 9, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2016Group: Database references
Revision 1.2Jan 10, 2024Group: 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase
D: 2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase
G: 2-hydroxymuconate tautomerase
H: 2-hydroxymuconate tautomerase
I: 2-hydroxymuconate tautomerase
J: 2-hydroxymuconate tautomerase
K: 2-hydroxymuconate tautomerase
L: 2-hydroxymuconate tautomerase
M: 2-hydroxymuconate tautomerase
N: 2-hydroxymuconate tautomerase
O: 2-hydroxymuconate tautomerase
P: 2-hydroxymuconate tautomerase
Q: 2-hydroxymuconate tautomerase
R: 2-hydroxymuconate tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,70519
Polymers114,55618
Non-polymers1491
Water4,396244
1
A: 2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase
D: 2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)38,1856
Polymers38,1856
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12920 Å2
ΔGint-56 kcal/mol
Surface area13690 Å2
MethodPISA
2
G: 2-hydroxymuconate tautomerase
H: 2-hydroxymuconate tautomerase
I: 2-hydroxymuconate tautomerase
J: 2-hydroxymuconate tautomerase
K: 2-hydroxymuconate tautomerase
L: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)38,1856
Polymers38,1856
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13160 Å2
ΔGint-56 kcal/mol
Surface area13970 Å2
MethodPISA
3
M: 2-hydroxymuconate tautomerase
N: 2-hydroxymuconate tautomerase
O: 2-hydroxymuconate tautomerase
P: 2-hydroxymuconate tautomerase
Q: 2-hydroxymuconate tautomerase
R: 2-hydroxymuconate tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,3347
Polymers38,1856
Non-polymers1491
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13130 Å2
ΔGint-59 kcal/mol
Surface area13780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.720, 61.166, 75.826
Angle α, β, γ (deg.)98.290, 95.160, 91.180
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C
41chain D
51chain E
61chain F
71chain G
81chain H
91chain I
101chain J
111chain K
121chain L
131chain M
141chain N
151chain O
161chain P
171chain Q
181chain R

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA1 - 57
211chain BB1 - 57
311chain CC1 - 54
411chain DD1 - 57
511chain EE1 - 57
611chain FF1 - 57
711chain GG1 - 57
811chain HH1 - 57
911chain II1 - 57
1011chain JJ1 - 59
1111chain KK1 - 57
1211chain LL1 - 59
1311chain MM1 - 57
1411chain NN1 - 57
1511chain OO1 - 57
1611chain PP1 - 57
1711chain QQ1 - 56
1811chain RR1 - 57

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Components

#1: Protein
2-hydroxymuconate tautomerase / / 4-oxalocrotonate tautomerase / 4-OT


Mass: 6364.202 Da / Num. of mol.: 18 / Fragment: UNP residues 2-60 / Mutation: M45Y, F50A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: xylH / Plasmid: pJexpress 414 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q01468, 2-hydroxymuconate tautomerase
#2: Chemical ChemComp-NS8 / trans beta nitrostyrene / Beta-Nitrostyrene


Mass: 149.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H7NO2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 244 / 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: 2.26 Å3/Da / Density % sol: 45.62 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.1 M PCTP buffer, 25% PEG 1500, 5 mM nitrostyrene, 5 % (v/v) DMSO

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: ROTATING ANODE / Type: BRUKER AXS MICROSTAR / Wavelength: 1.5418 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jan 17, 2015 / Details: HELIOS OPTICS
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.3→56.4 Å / Num. all: 86455 / Num. obs: 42246 / % possible obs: 94.7 % / Redundancy: 2.05 % / Rmerge(I) obs: 0.074 / Net I/σ(I): 10.7
Reflection shellResolution: 2.3→2.38 Å / Rmerge(I) obs: 0.414 / Mean I/σ(I) obs: 2 / % possible all: 93.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
Aimlessdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5CLN

5cln


Resolution: 2.3→43.965 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 28.8 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2764 2004 4.76 %
Rwork0.2346 40116 -
obs0.2366 42120 94.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 80 Å2 / Biso mean: 32.5738 Å2 / Biso min: 6.8 Å2
Refinement stepCycle: final / Resolution: 2.3→43.965 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7761 0 22 244 8027
Biso mean--41.84 28.87 -
Num. residues----1026
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0047855
X-RAY DIFFRACTIONf_angle_d0.87610575
X-RAY DIFFRACTIONf_chiral_restr0.0321277
X-RAY DIFFRACTIONf_plane_restr0.0031355
X-RAY DIFFRACTIONf_dihedral_angle_d14.4972975
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4827X-RAY DIFFRACTION8.048TORSIONAL
12B4827X-RAY DIFFRACTION8.048TORSIONAL
13C4827X-RAY DIFFRACTION8.048TORSIONAL
14D4827X-RAY DIFFRACTION8.048TORSIONAL
15E4827X-RAY DIFFRACTION8.048TORSIONAL
16F4827X-RAY DIFFRACTION8.048TORSIONAL
17G4827X-RAY DIFFRACTION8.048TORSIONAL
18H4827X-RAY DIFFRACTION8.048TORSIONAL
19I4827X-RAY DIFFRACTION8.048TORSIONAL
110J4827X-RAY DIFFRACTION8.048TORSIONAL
111K4827X-RAY DIFFRACTION8.048TORSIONAL
112L4827X-RAY DIFFRACTION8.048TORSIONAL
113M4827X-RAY DIFFRACTION8.048TORSIONAL
114N4827X-RAY DIFFRACTION8.048TORSIONAL
115O4827X-RAY DIFFRACTION8.048TORSIONAL
116P4827X-RAY DIFFRACTION8.048TORSIONAL
117Q4827X-RAY DIFFRACTION8.048TORSIONAL
118R4827X-RAY DIFFRACTION8.048TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.3001-2.35760.35391370.29612875301293
2.3576-2.42130.32851480.26862832298094
2.4213-2.49260.31731300.25822886301695
2.4926-2.5730.30611510.24862890304195
2.573-2.66490.31781430.25122880302395
2.6649-2.77160.28831520.2422887303996
2.7716-2.89770.32111530.24682905305896
2.8977-3.05050.32311330.26762918305196
3.0505-3.24160.29191530.25692934308797
3.2416-3.49170.30391330.23762933306697
3.4917-3.84290.29381190.26072278239775
3.8429-4.39860.25961560.22792942309897
4.3986-5.540.22421390.1923004314399
5.54-43.97270.20041570.18772952310998

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