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- PDB-6ach: Structure of NAD+-bound leucine dehydrogenase from Geobacillus st... -

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Basic information

Entry
Database: PDB / ID: 6ach
TitleStructure of NAD+-bound leucine dehydrogenase from Geobacillus stearothermophilus by cryo-EM
ComponentsLeucine dehydrogenase
KeywordsOXIDOREDUCTASE / LEUCINE DEHYDROGENSE / NAD/LEUCINE BINDING / HOLO 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 / 3.2 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)329,98016
Polyers324,6728
Non-polymers5,3078
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)42120
ΔGint (kcal/M)-53
Surface area (Å2)111640
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
#2: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 8 / Formula: C21H27N7O14P2 / Nicotinamide adenine dinucleotide / Comment: NAD *YM

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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: Binary complex of leucine dehydrogenase with NAD+ / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.305 kDa/nm / 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
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
IDNameVersionCategoryDetails
1EMAN2particle selection
4CTFFINDCTF correction3
9PHENIX1.12model refinement
10EMAN2initial Euler assignment
11RELION2.0.3final Euler assignment
12RELION2.0.3classification
13RELION2.0.33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: D4
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 92034 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01323688
ELECTRON MICROSCOPYf_angle_d1.14832024
ELECTRON MICROSCOPYf_dihedral_angle_d7.64719496
ELECTRON MICROSCOPYf_chiral_restr0.0673448
ELECTRON MICROSCOPYf_plane_restr0.0074200

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