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- PDB-8xcy: Cryo-EM structure of Glutamate dehydrogenase from Thermococcus pr... -

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Basic information

Entry
Database: PDB / ID: 8xcy
TitleCryo-EM structure of Glutamate dehydrogenase from Thermococcus profundus incorporating NADP and GLU in the steady stage of reaction
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE / Complex / Coenzyme / NADP / Glutamate
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
GAMMA-L-GLUTAMIC ACID / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsWakabayashi, T. / Oide, M. / Nakasako, M.
Funding support Japan, 14items
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)jp18J11653 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
Japan Science and TechnologyJPMJPR22E2 Japan
CitationJournal: Sci Rep / Year: 2024
Title: CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase.
Authors: Taiki Wakabayashi / Mao Oide / Masayoshi Nakasako /
Abstract: Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic ...Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.
History
DepositionDec 10, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,6493
Polymers46,7581
Non-polymers8912
Water1448
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


Mass: 46758.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus profundus (archaea) / Gene: gdhA / Production host: Escherichia coli (E. coli)
References: UniProt: O74024, glutamate dehydrogenase [NAD(P)+]
#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GGL / GAMMA-L-GLUTAMIC ACID / L-GLUTAMIC ACID


Type: L-gamma-peptide, C-delta linking / Mass: 147.129 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H9NO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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 glutamate dehydrogenase in the presence of NADP and glutamate
Type: COMPLEX / 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
Buffer component
IDConc.NameFormulaBuffer-ID
10.5 mMnicotinamide adenine dinucleotide phosphateNADP1
2100 mMglutamateGlu1
35 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenConc.: 4.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: The sample solution kept at room temperature was flash-frozen 1-h after mixing GDH, NADP and glutamate solutions.

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Details: Grid information as following: Company/model: Quantifoil Cu 1.2/1.3 Material:Cu Grid mesh: 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 47000 nm / Nominal defocus min: 400 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7075

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Processing

EM software
IDNameVersionCategory
1RELION4.0betaparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION4.0betainitial Euler assignment
11RELION4.0betafinal Euler assignment
12RELION4.0betaclassification
13RELION4.0beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6334528
Details: Auto-picking based on templates from manually picked GDH particles.
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86791 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033299
ELECTRON MICROSCOPYf_angle_d0.4834481
ELECTRON MICROSCOPYf_dihedral_angle_d3.689450
ELECTRON MICROSCOPYf_chiral_restr0.043493
ELECTRON MICROSCOPYf_plane_restr0.004576

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