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- PDB-6acf: structure of leucine dehydrogenase from Geobacillus stearothermop... -

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Basic information

Entry
Database: PDB / ID: 6acf
Titlestructure of leucine dehydrogenase from Geobacillus stearothermophilus by cryo-EM
ComponentsLeucine dehydrogenase
KeywordsOXIDOREDUCTASE / LEUCINE DEHYDROGENSE / NAD/LEUCINE BINDING / APO FORM
Function / homologyGlutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase, bacterial/archaeal / Leu/Phe/Val dehydrogenases active site / NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glu / Leu / Phe / Val dehydrogenases active site. / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor ...Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase, bacterial/archaeal / Leu/Phe/Val dehydrogenases active site / NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glu / Leu / Phe / Val dehydrogenases active site. / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor / cellular amino acid metabolic process / nucleotide binding / Leucine dehydrogenase
Function and homology information
Specimen sourceGeobacillus stearothermophilus 10 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3 Å resolution
AuthorsYamaguchi, H. / Kamegawa, A. / Nakata, K. / Kashiwagi, T. / Mizukoshi, T. / Fujiyoshi, Y. / Tani, K.
CitationJournal: J. Struct. Biol. / Year: 2018
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 26, 2018 / Release: Dec 26, 2018

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Assembly

Deposited unit
A: Leucine dehydrogenase
B: Leucine dehydrogenase
C: Leucine dehydrogenase
D: Leucine dehydrogenase
E: Leucine dehydrogenase
F: Leucine dehydrogenase
G: Leucine dehydrogenase
H: Leucine dehydrogenase


Theoretical massNumber of molelcules
Total (without water)324,6728
Polyers324,6728
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)34930
ΔGint (kcal/M)-20
Surface area (Å2)112570
MethodPISA

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Components

#1: Protein/peptide
Leucine dehydrogenase /


Mass: 40584.020 Da / Num. of mol.: 8
Source: (gene. exp.) Geobacillus stearothermophilus 10 (bacteria)
Gene: GT50_15010 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0K2HC96

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: The apo form of Leucine dehydrogenase / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.305 MDa / Experimental value: YES
Source (natural)Organism: Geobacillus stearothermophilus 10 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 10.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 200 / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyMicroscope model: JEOL KYOTO-3000SFF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm
Image recordingElectron dose: 2.7 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN2particle selection
4CTFFIND3CTF correction
9EMAN2initial Euler assignment
10RELION2.03final Euler assignment
11RELION2.0.3classification
12RELION2.0.33D reconstruction
13PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: D4
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 132800 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00823584
ELECTRON MICROSCOPYf_angle_d0.93531840
ELECTRON MICROSCOPYf_dihedral_angle_d8.84719632
ELECTRON MICROSCOPYf_chiral_restr0.0573416
ELECTRON MICROSCOPYf_plane_restr0.0074240

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