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- EMDB-60269: Cryo-EM structure of W89F mutated Glutamate dehydrogenase from Th... -

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Basic information

Entry
Database: EMDB / ID: EMD-60269
TitleCryo-EM structure of W89F mutated Glutamate dehydrogenase from Thermococcus profundus in complex with NADP and GLU in the steady stage of reaction
Map data
Sample
  • Complex: Hexamer of W89F mutated glutamate dehydrogenase
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: GLUTAMIC ACID
  • Ligand: water
KeywordsOxidoreductase / Complex / NADP / Glutamate / 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
Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsWakabayashi T / Nakasako M
Funding support Japan, 13 items
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, 2024-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60269.png

Downloads & links

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Map

FileDownload / File: emd_60269.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 256 pix.
= 192.512 Å		0.75 Å/pix.
x 256 pix.
= 192.512 Å		0.75 Å/pix.
x 256 pix.
= 192.512 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.752 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.022760842 - 0.039662164
Average (Standard dev.)0.000049763315 (±0.0019214286)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 192.512 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60269_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_60269_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60269_half_map_2.map
Projections & Slices
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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamer of W89F mutated glutamate dehydrogenase

EntireName: Hexamer of W89F mutated glutamate dehydrogenase
Components
  • Complex: Hexamer of W89F mutated glutamate dehydrogenase
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: GLUTAMIC ACID
  • Ligand: water

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Supramolecule #1: Hexamer of W89F mutated glutamate dehydrogenase

SupramoleculeName: Hexamer of W89F mutated glutamate dehydrogenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermococcus profundus (archaea)
Molecular weightTheoretical: 280 KDa

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Macromolecule #1: Glutamate dehydrogenase

MacromoleculeName: Glutamate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: glutamate dehydrogenase [NAD(P)+]
Source (natural)Organism: Thermococcus profundus (archaea)
Molecular weightTheoretical: 46.360004 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: IDPFEMAVKQ LERAAQYMDI SEEALEWLKK PMRIVEVSVP IEMDDGSVKV FTGFRVQHNW ARGPTKGGIR WHPAETLSTV KALATFMTW KVAVVDLPYG GGKGGIIVNP KELSEREQER LARAYIRAVY DVIGPWTDIP APDVYTNPKI MGWMMDEYET I MRRKGPAF ...String:
IDPFEMAVKQ LERAAQYMDI SEEALEWLKK PMRIVEVSVP IEMDDGSVKV FTGFRVQHNW ARGPTKGGIR WHPAETLSTV KALATFMTW KVAVVDLPYG GGKGGIIVNP KELSEREQER LARAYIRAVY DVIGPWTDIP APDVYTNPKI MGWMMDEYET I MRRKGPAF GVITGKPLSI GGSLGRGTAT AQGAIFTIRE AAKALGIDLK GKKIAVQGYG NAGYYTAKLA KEQLGMTVVA VS DSRGGIY NPDGLDPDEV LKWKREHGSV KDFPGATNIT NEELLELEVD VLAPAAIEEV ITEKNADNIK AKIVAEVANG PVT PEADDI LREKGILQIP DFLCNAGGVT VSYFEWVQNI NGYYWTEEEV REKLDKKMTK AFWEVYNTHK DKNIHMRDAA YVVA VSRVY QAMKDRGWVK K

UniProtKB: Glutamate dehydrogenase

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Macromolecule #2: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #3: GLUTAMIC ACID

MacromoleculeName: GLUTAMIC ACID / type: ligand / ID: 3 / Number of copies: 1 / Formula: GLU
Molecular weightTheoretical: 147.129 Da
Chemical component information

ChemComp-GLU:
GLUTAMIC ACID

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 9 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration9.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.5 mMC21H27N7NaO17P3Sodium Nicotiamide adenine dinucleotide phosphate
100.0 mMC5H8NNaO4monosodium glutamate
5.0 mMC4H11NO3tris(hydroxymethyl)aminomethane

Details: 0.5 mM NADP, 100 mM Glutamate in 5 mM Tris-HCl at pH7.5.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa
Details: Both sides of the grid were glow-discharged for 45 s at 20 mA and 20 Pa.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: The sample solution was flash-frozen 2-h after mixing the protein solution and buffer solution..
Details8.96 mg/mL GDH W89F

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7651 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.35000000000000003 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 3534400
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Details: The startup model was generated by de novo 3D model generation in Relion.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 119274
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 32 / Avg.num./class: 115000 / Software - Name: RELION (ver. 4.0)
Details: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into ...Details: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into four subclasses. This entire process was conducted following a symmetry expansion operation on all particles, in accordance with the D3 symmetry of GDH.
FSC plot (resolution estimation)

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