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- PDB-8znc: Cryo-EM structure of W89F mutated Glutamate dehydrogenase from Th... -

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Basic information

Entry
Database: PDB / ID: 8znc
TitleCryo-EM structure of W89F mutated Glutamate dehydrogenase from Thermococcus profundus incorporating NADPH, AKG in the steady stage of reaction
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE / Complex / NADPH / 2-oxoglutarate / Mutant
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
2-OXOGLUTARIC ACID / Chem-NDP / AMMONIUM ION / Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å
AuthorsWakabayashi, T. / Nakasako, 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 26, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 12, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / 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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,2704
Polymers46,3601
Non-polymers9103
Water43224
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Glutamate dehydrogenase / GDH


Mass: 46360.004 Da / Num. of mol.: 1 / 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)+]
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#3: Chemical ChemComp-AKG / 2-OXOGLUTARIC ACID


Mass: 146.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H6O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-NH4 / AMMONIUM ION


Mass: 18.038 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H4N / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hexamer of W89F mutated glutamate dehydrogenase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.280 MDa / Experimental value: YES
Source (natural)Organism: Thermococcus profundus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 0.5 mM NADP, 100 mM Glutamate in 5 mM Tris-HCl at pH7.5.
Buffer component
IDConc.NameFormulaBuffer-ID
10.5 mMSodium Nicotiamide adenine dinucleotide phosphateC21H27N7NaO17P31
2100 mMmonosodium glutamateC5H8NNaO41
35 mMtris(hydroxymethyl)aminomethaneC4H11NO31
SpecimenConc.: 9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 8.96 mg/mL GDH W89F
Specimen supportDetails: Both sides of the grid were glow-discharged for 45 s at 20 mA and 20 Pa.
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: The sample solution was flash-frozen 2-h after mixing the protein solution and buffer solution.

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 350 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7651

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
2SerialEMimage acquisition
4CTFFIND4.1.14CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3534400
3D reconstructionResolution: 2.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 444903 / Num. of class averages: 1 / Symmetry type: POINT

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