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- PDB-4c9s: BACTERIAL CHALCONE ISOMERASE IN open CONFORMATION FROM EUBACTERIU... -

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Basic information

Entry
Database: PDB / ID: 4c9s
TitleBACTERIAL CHALCONE ISOMERASE IN open CONFORMATION FROM EUBACTERIUM RAMULUS AT 1.8 A RESOLUTION
ComponentsCHALCONE ISOMERASE
KeywordsISOMERASE / FLAVONOIDS
Function / homologyChalcone isomerase, N-terminal / Chalcone isomerase N-terminal domain / chalcone isomerase / chalcone isomerase activity / Chalcone isomerase
Function and homology information
Biological speciesEUBACTERIUM RAMULUS (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / Resolution: 1.8 Å
AuthorsThomsen, M. / Palm, G.J. / Hinrichs, W.
Citation
Journal: Acta Crystallogr D Biol Crystallogr / Year: 2015
Title: Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.
Authors: Maren Thomsen / Anne Tuukkanen / Jonathan Dickerhoff / Gottfried J Palm / Hanna Kratzat / Dmitri I Svergun / Klaus Weisz / Uwe T Bornscheuer / Winfried Hinrichs /
Abstract: Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins ...Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.
#1: Journal: Arch.Microbiol. / Year: 2004
Title: First Bacterial Chalcone Isomerase Isolated from Eubacterium Ramulus.
Authors: Herles, C. / Braune, A. / Blaut, M.
History
DepositionOct 3, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 22, 2014Provider: repository / Type: Initial release
Revision 1.1Apr 8, 2015Group: Database references
Revision 1.2Apr 22, 2015Group: Database references
Revision 1.3Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CHALCONE ISOMERASE
B: CHALCONE ISOMERASE
C: CHALCONE ISOMERASE
D: CHALCONE ISOMERASE
E: CHALCONE ISOMERASE
F: CHALCONE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)195,72731
Polymers194,4406
Non-polymers1,28725
Water45,0382500
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31900 Å2
ΔGint-303.6 kcal/mol
Surface area52350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)171.246, 192.290, 204.635
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number24
Space group name H-MI212121

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Components

#1: Protein
CHALCONE ISOMERASE


Mass: 32406.736 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) EUBACTERIUM RAMULUS (bacteria) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA / References: UniProt: V9P0A9*PLUS, chalcone isomerase
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2500 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsGENBANK KF154734

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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: 4.65 Å3/Da / Density % sol: 73.6 % / Description: NONE
Crystal growpH: 7.5
Details: 0.1 M HEPES PH 7.5, 0.2 M SODIUM CHLORIDE, 1.7 M AMMONIUM SULFATE; CRYO: 0.1 M HEPES PH 7.5, 0.2 M SODIUM CHLORIDE, 2 M AMMONIUM SULFATE, 22% GLYCEROL

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.97935
DetectorType: MARRESEARCH / Detector: CCD / Date: Nov 28, 2012 / Details: MIRROR
RadiationMonochromator: MIRROR / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97935 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. obs: 309268 / % possible obs: 99.6 % / Observed criterion σ(I): 2 / Redundancy: 4.15 % / Biso Wilson estimate: 26.6 Å2 / Rmerge(I) obs: 0.1 / Net I/σ(I): 12.25
Reflection shellResolution: 1.8→1.91 Å / Redundancy: 4.08 % / Rmerge(I) obs: 0.7 / Mean I/σ(I) obs: 2.14 / % possible all: 97.9

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Processing

Software
NameVersionClassification
REFMAC5.7.0027refinement
XDSdata reduction
XDSdata scaling
SHELXCDEphasing
RefinementMethod to determine structure: SIRAS
Starting model: PDB ENTRY 4C9T

4c9t


Resolution: 1.8→48.07 Å / Cor.coef. Fo:Fc: 0.975 / Cor.coef. Fo:Fc free: 0.968 / SU B: 3.048 / SU ML: 0.042 / Cross valid method: THROUGHOUT / ESU R: 0.064 / ESU R Free: 0.065 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.15389 15464 5 %RANDOM
Rwork0.13446 ---
obs0.13544 293803 99.57 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 20.077 Å2
Baniso -1Baniso -2Baniso -3
1--0.61 Å20 Å20 Å2
2--0.7 Å20 Å2
3----0.1 Å2
Refinement stepCycle: LAST / Resolution: 1.8→48.07 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12791 0 59 2500 15350
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0213514
X-RAY DIFFRACTIONr_bond_other_d0.0010.0212429
X-RAY DIFFRACTIONr_angle_refined_deg1.7141.94218424
X-RAY DIFFRACTIONr_angle_other_deg0.845328707
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.62551611
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.99123.963656
X-RAY DIFFRACTIONr_dihedral_angle_3_deg10.766152171
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.5431566
X-RAY DIFFRACTIONr_chiral_restr0.1230.21869
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.02115297
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023245
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.797→1.844 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.248 1088 -
Rwork0.227 20672 -
obs--95.42 %
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)
10.10580.05680.0510.1828-0.0650.14720.0181-0.00460.03070.0661-0.00650.0147-0.00570.0023-0.01150.12990.01260.01670.0383-0.00980.056109.453131.59457.004
20.2581-0.05520.07070.09540.02930.068-0.0026-0.0213-0.01390.00720.02370.03840.01590.0055-0.02110.0970.00430.01430.03260.02510.088989.307114.25138.822
30.1106-0.11980.00740.1556-0.01410.0234-0.0119-0.00910.020.01830.01170.0055-0.00610.03440.00020.07080.01470.01450.0748-0.00540.0678140.658117.44435.597
40.0878-0.0037-0.04830.073-0.04730.0686-0.02690.0182-0.0268-0.003-0.00440.00360.01740.00660.03130.08020.0170.0160.0637-0.00450.0768120.521100.8116.774
50.0273-0.00860.01510.1131-0.0160.0420.0001-0.00220.00310.0212-0.00710.00670.01170.03870.0070.04520.001-0.00490.0555-0.00110.061122.263151.73224.386
60.0304-0.0263-0.00180.2045-0.06810.08830.01330.0260.0104-0.01770.01160.03910.0033-0.0116-0.02490.10160.02460.00660.04960.01090.0775101.707134.9626.239
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 105
2X-RAY DIFFRACTION1A106 - 130
3X-RAY DIFFRACTION1A131 - 282
4X-RAY DIFFRACTION2B1 - 105
5X-RAY DIFFRACTION2B106 - 130
6X-RAY DIFFRACTION2B131 - 282
7X-RAY DIFFRACTION3C1 - 105
8X-RAY DIFFRACTION3C106 - 130
9X-RAY DIFFRACTION3C131 - 282
10X-RAY DIFFRACTION4D1 - 105
11X-RAY DIFFRACTION4D106 - 130
12X-RAY DIFFRACTION4D131 - 282
13X-RAY DIFFRACTION5E1 - 105
14X-RAY DIFFRACTION5E106 - 130
15X-RAY DIFFRACTION5E131 - 282
16X-RAY DIFFRACTION6F1 - 105
17X-RAY DIFFRACTION6F106 - 130
18X-RAY DIFFRACTION6F131 - 282

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