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- EMDB-9592: Structure of NAD+-bound leucine dehydrogenase from Geobacillus st... -

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Basic information

Entry
Database: EMDB / ID: EMD-9592
TitleStructure of NAD+-bound leucine dehydrogenase from Geobacillus stearothermophilus by cryo-EM
Map dataThe holo form of GstLDH
Sample
  • Complex: Binary complex of leucine dehydrogenase with NAD+
    • Protein or peptide: Leucine dehydrogenase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
KeywordsLEUCINE DEHYDROGENSE / NAD/LEUCINE BINDING / HOLO FORM / OXIDOREDUCTASE
Function / homology
Function and homology information


oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor / amino acid metabolic process / nucleotide binding
Similarity search - Function
Glutamate/phenylalanine/leucine/valine dehydrogenase, bacterial/archaeal / 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 / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Leucine dehydrogenase
Similarity search - Component
Biological speciesGeobacillus stearothermophilus 10 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYamaguchi H / Kamegawa A / Nakata K / Kashiwagi T / Mizukoshi T / Fujiyoshi Y / Tani K
CitationJournal: J Struct Biol / Year: 2019
Title: Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.
Authors: Hiroki Yamaguchi / Akiko Kamegawa / Kunio Nakata / Tatsuki Kashiwagi / Toshimi Mizukoshi / Yoshinori Fujiyoshi / Kazutoshi Tani /
Abstract: Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a ...Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD-bound LDH are reported at 3.0 and 3.2 Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD. NAD binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.
History
DepositionJul 26, 2018-
Header (metadata) releaseDec 26, 2018-
Map releaseDec 26, 2018-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6ach
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9592.png

Downloads & links

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Map

FileDownload / File: emd_9592.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe holo form of GstLDH
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 192 pix.
= 236.544 Å		1.23 Å/pix.
x 192 pix.
= 236.544 Å		1.23 Å/pix.
x 192 pix.
= 236.544 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.232 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.22664541 - 0.4768642
Average (Standard dev.)0.000025061747 (±0.016380014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 236.544 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2321.2321.232
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.544236.544236.544
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2270.4770.000

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Supplemental data

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Sample components

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Entire : Binary complex of leucine dehydrogenase with NAD+

EntireName: Binary complex of leucine dehydrogenase with NAD+
Components
  • Complex: Binary complex of leucine dehydrogenase with NAD+
    • Protein or peptide: Leucine dehydrogenase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: Binary complex of leucine dehydrogenase with NAD+

SupramoleculeName: Binary complex of leucine dehydrogenase with NAD+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Geobacillus stearothermophilus 10 (bacteria)
Molecular weightTheoretical: 0.305 kDa/nm

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Macromolecule #1: Leucine dehydrogenase

MacromoleculeName: Leucine dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus 10 (bacteria)
Molecular weightTheoretical: 40.58402 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MELFQYMEKY DYEQVLFCQD KESGLKAIIV IHDTTLGPAL GGTRMWMYNS EEEALEDALR LARGMTYKNA AAGLNLGGGK TVIIGDPRK DKNEAMFRAF GRFIQGLNGR YITAEDVGTT VADMDIIYQE TDYVTGISPE FGSSGNPSPA TAYGVYRGMK A AAKEAFGS ...String:
MELFQYMEKY DYEQVLFCQD KESGLKAIIV IHDTTLGPAL GGTRMWMYNS EEEALEDALR LARGMTYKNA AAGLNLGGGK TVIIGDPRK DKNEAMFRAF GRFIQGLNGR YITAEDVGTT VADMDIIYQE TDYVTGISPE FGSSGNPSPA TAYGVYRGMK A AAKEAFGS DSLEGKVVAV QGVGNVAYHL CRHLHEEGAK LIVTDINKEA VARAVEEFGA KAVDPNDIYG VECDIFAPCA LG GIINDQT IPQLKAKVIA GSANNQLKEP RHGDMIHEMG IVYAPDYVIN AGGVINVADE LYGYNRERAM KKIEQIYDNI EKV FAIAKR DNIPTYVAAD RMAEERIETM RKARSQFLQN GHHILSRRRA R

UniProtKB: Leucine dehydrogenase

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Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 8 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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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

Concentration0.5 mg/mL
BufferpH: 10.5
GridModel: Quantifoil R2/2 / Material: MOLYBDENUM / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 2.7 e/Å2

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: EMAN2
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 2.0.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0.3)
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0.3) / Number images used: 92034
FSC plot (resolution estimation)

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