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- PDB-8zmu: GLUTAMATE DEHYDROGENASE (W89F-MUTANT) FROM THERMOCOCCUS PROFUNDUS... -

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Basic information

Entry
Database: PDB / ID: 8zmu
TitleGLUTAMATE DEHYDROGENASE (W89F-MUTANT) FROM THERMOCOCCUS PROFUNDUS IN THE UNLIGANDED STATE
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE
Function / homology
Function and homology information


glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NAD+) activity / glutamate dehydrogenase (NADP+) activity / L-glutamate catabolic process
Similarity search - Function
: / Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal ...: / Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Aminoacid dehydrogenase-like, N-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
ACETIC ACID / Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.028 Å
AuthorsWakabayashi, T. / Matsui, Y. / Masayoshi, M.
Funding support Japan, 13items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)jp13480214 Japan
Japan Society for the Promotion of Science (JSPS)jp19204042 Japan
Japan Society for the Promotion of Science (JSPS)jp22244054 Japan
Japan Society for the Promotion of Science (JSPS)jp21H01050 Japan
Japan Society for the Promotion of Science (JSPS)jp26800227 Japan
Japan Society for the Promotion of Science (JSPS)18J11653 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15076210 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp20050030 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp22018027 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp23120525 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp25120725 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15H01647 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp17H05891 Japan
CitationJournal: FEBS J / Year: 2025
Title: CryoEM and crystal structure analyses reveal the indirect role played by Trp89 in glutamate dehydrogenase enzymatic reactions.
Authors: Taiki Wakabayashi / Yuka Matsui / Masayoshi Nakasako /
Abstract: Glutamate dehydrogenase from Thermococcus profundus is a homo-hexameric enzyme that catalyzes the reversible deamination of glutamate to 2-oxoglutarate in the presence of a cofactor. In each subunit, ...Glutamate dehydrogenase from Thermococcus profundus is a homo-hexameric enzyme that catalyzes the reversible deamination of glutamate to 2-oxoglutarate in the presence of a cofactor. In each subunit, a large active-site cleft is formed between the two functional domains, one of which displays motion to open and close the cleft. Trp89 in the cleft displays two sidechain conformers in the open cleft and a single conformer in the closed cleft. To reveal the role of the Trp89 sidechain in the domain motion, we mutated Trp89 to phenylalanine. Despite the Trp89 sidechain being located away from the reaction center, the catalytic constant decreased to 1/38-fold of that of the wild-type without a fatal reduction of the affinities to the cofactor and ligand molecules. To understand the molecular mechanism underlying this reduction, we determined the crystal structure in the unliganded state and the metastable conformations appearing in the steady stage of the reaction using cryo-electron microscopy (cryoEM). The four identified metastable conformations were similar to the three conformations observed in the wild-type, but their populations were different from those of the wild-type. In addition, a conformation with a completely closed active-site cleft necessary for the reaction to proceed was quite rare. The crystal structure and the four metastable conformations suggested that the reduction in the catalytic constant could be attributed to changes in the interactions between Gln13 and the 89th side chains, preventing the closing domain motion.
History
DepositionMay 23, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate dehydrogenase
B: Glutamate dehydrogenase
C: Glutamate dehydrogenase
D: Glutamate dehydrogenase
E: Glutamate dehydrogenase
F: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,87268
Polymers280,3176
Non-polymers5,55562
Water17,078948
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)113.355, 163.248, 132.605
Angle α, β, γ (deg.)90.00, 113.91, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
Glutamate dehydrogenase / GDH


Mass: 46719.441 Da / Num. of mol.: 6 / Mutation: W89F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus profundus (archaea) / Gene: gdhA, gdh / Production host: Escherichia coli (E. coli)
References: UniProt: O74024, glutamate dehydrogenase [NAD(P)+]

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Non-polymers , 5 types, 1010 molecules

#2: Chemical...
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 43 / Source method: isolated from a natural source / Formula: SO4
#3: Chemical
ChemComp-ACY / ACETIC ACID


Mass: 60.052 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: C2H4O2
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: C3H8O3
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Na
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 948 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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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: 4.04 Å3/Da / Density % sol: 69.58 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 3.8
Details: 1.6 M lithium sulfate (Wako, Osaka, Japan), 1.7%(w/v) poly-ethylene glycol 8000 (PEG800) (Sigma-Aldrich, St. Louis, USA) and 0.1 M sodium acetate (Wako)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL26B2 / Wavelength: 1 Å
DetectorType: RAYONIX SX-165mm / Detector: CCD / Date: Oct 9, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.028→50 Å / Num. obs: 283125 / % possible obs: 99.8 % / Redundancy: 3.788 % / Rmerge(I) obs: 0.063 / Net I/σ(I): 18.3
Reflection shellResolution: 2.028→2.07 Å / Rmerge(I) obs: 0.293 / Num. unique obs: 281936

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.028→21.8 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.949 / SU B: 3.285 / SU ML: 0.087 / Cross valid method: THROUGHOUT / ESU R: 0.119 / ESU R Free: 0.115 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.19944 14013 5 %RANDOM
Rwork0.17224 ---
obs0.17357 269065 99.58 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 39.29 Å2
Baniso -1Baniso -2Baniso -3
1--1.27 Å20 Å21.54 Å2
2---1.23 Å2-0 Å2
3---0.81 Å2
Refinement stepCycle: 1 / Resolution: 2.028→21.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19378 0 308 948 20634
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.01320220
X-RAY DIFFRACTIONr_bond_other_d0.0010.01719305
X-RAY DIFFRACTIONr_angle_refined_deg1.7661.64427405
X-RAY DIFFRACTIONr_angle_other_deg1.4511.58444497
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.96652492
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.12822.566990
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.719153483
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.03715121
X-RAY DIFFRACTIONr_chiral_restr0.090.22582
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0222548
X-RAY DIFFRACTIONr_gen_planes_other0.0010.024410
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_it3.793.7749985
X-RAY DIFFRACTIONr_mcbond_other3.7853.7739976
X-RAY DIFFRACTIONr_mcangle_it5.1075.64312463
X-RAY DIFFRACTIONr_mcangle_other5.1075.64312464
X-RAY DIFFRACTIONr_scbond_it5.3994.43910235
X-RAY DIFFRACTIONr_scbond_other5.3994.43910236
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other8.0486.4114942
X-RAY DIFFRACTIONr_long_range_B_refined9.40544.77622967
X-RAY DIFFRACTIONr_long_range_B_other9.40544.77722968
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.028→2.081 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.258 1054 -
Rwork0.238 19369 -
obs--97.41 %

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