|Entry||Database: PDB / ID: 6acf|
|Title||structure of leucine dehydrogenase from Geobacillus stearothermophilus by cryo-EM|
|Keywords||OXIDOREDUCTASE / LEUCINE DEHYDROGENSE / NAD/LEUCINE BINDING / APO FORM|
|Function / homology||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 ...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 source||Geobacillus stearothermophilus 10 (bacteria)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3 Å resolution|
|Authors||Yamaguchi, H. / Kamegawa, A. / Nakata, K. / Kashiwagi, T. / Mizukoshi, T. / Fujiyoshi, Y. / Tani, K.|
|Citation||Journal: 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 18.104.22.168) 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 22.214.171.124) 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.
SummaryFull reportAbout validation report
|Date||Deposition: Jul 26, 2018 / Release: Dec 26, 2018|
|Structure viewer||Molecule: |
Downloads & links
A: Leucine dehydrogenase
B: Leucine dehydrogenase
C: Leucine dehydrogenase
D: Leucine dehydrogenase
E: Leucine dehydrogenase
F: Leucine dehydrogenase
G: Leucine dehydrogenase
H: 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
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: The apo form of Leucine dehydrogenase / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.305 MDa / Experimental value: YES|
|Source (natural)||Organism: Geobacillus stearothermophilus 10 (bacteria)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 10.5|
|Specimen||Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: MOLYBDENUM / Grid mesh size: 200 / Grid type: Quantifoil R2/2|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
|Microscopy||Microscope model: JEOL KYOTO-3000SFF|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm|
|Image recording||Electron dose: 2.7 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|Software||Name: PHENIX / Version: 1.12_2829: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING ONLY|
|Symmetry||Point symmetry: D4|
|3D reconstruction||Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 132800 / Symmetry type: POINT|
|Refine LS restraints|
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