PDBsum entry 1uxi

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Oxidoreductase PDB id
Protein chains
308 a.a. *
NAD ×2
FUM ×2
Waters ×450
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface
Structure: Malate dehydrogenase. Chain: a, b. Engineered: yes. Mutation: yes
Source: Chloroflexus aurantiacus. Organism_taxid: 1108. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Tetramer (from PDB file)
2.10Å     R-factor:   0.243     R-free:   0.276
Authors: A.Bjork,B.Dalhus,D.Mantzilas,V.G.H.Eijsink,R.Sirevag
Key ref:
A.Bjørk et al. (2004). Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface. J Mol Biol, 341, 1215-1226. PubMed id: 15321717 DOI: 10.1016/j.jmb.2004.06.079
25-Feb-04     Release date:   26-Aug-04    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P80040  (MDH_CHLAA) -  Malate dehydrogenase
309 a.a.
308 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Malate dehydrogenase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Citric acid cycle
      Reaction: (S)-malate + NAD+ = oxaloacetate + NADH
Bound ligand (Het Group name = FUM)
matches with 88.89% similarity
Bound ligand (Het Group name = NAD)
corresponds exactly
= oxaloacetate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   5 terms 
  Biochemical function     catalytic activity     4 terms  


DOI no: 10.1016/j.jmb.2004.06.079 J Mol Biol 341:1215-1226 (2004)
PubMed id: 15321717  
Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.
A.Bjørk, B.Dalhus, D.Mantzilas, R.Sirevåg, V.G.Eijsink.
The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.
  Selected figure(s)  
Figure 1.
Figure 1. The structure of homotetrameric MDH from C. aurantiacus is shown in a ribbon diagram in the upper panel. The tetramer consists of two dimers (AB and CD). The NAD cofactor bound to the catalytic centre is shown in yellow. The rectangle indicates the location of the electrostatic network around Glu165 at the dimer--dimer interface in CaMDH (note that the network occurs four times in the tetramer). The lower panel shows a stereo view of the environment of Glu165 in subunit A, with backbones coloured according to the scheme used in the upper panel. The side-chains are shown in ball and stick in atom colours, Cd 2C bound to Glu165 is shown in yellow. Dotted lines indicate interacting atoms, located within 3 Å of each other.
Figure 5.
Figure 5. Stereo pictures showing the environment of residue 165 in cadmium-containing structures of wild-type CaMDH (A) and the E165Q (B) and E165K (C) mutants. (B) and (C) show 2F oKFc maps contoured at 1s for residue 165 to illustrate the conformation of the side-chains as well as the general quality of the refinement. Red spheres represent water molecules; the yellow sphere represents cadmium. In CaMDH (A) the negatively charged Glu165 binds directly to one of the cadmium ions in the structure (closest distance 2.5 A ). In the E165Q mutant (B) the cadmium ion binds to residue 165 via a water molecule (closest Cd 2C -165 distance 4.2 A ). In the E165K mutant (A) the cadmium ion has moved even further away from its original position and is now very close (2.3 A , as compared to 4.2 A in the wild-type) to Glu238 of the other monomer. The distance between cadmium and the z-amino group of Lys165 is 4.1 A . Figure 1 and this Figure were made using the programs Bobscript 64 and Raster 3D. 65
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 341, 1215-1226) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19220855 G.A.Kotzia, and N.E.Labrou (2009).
Engineering thermal stability of L-asparaginase by in vitro directed evolution.
  FEBS J, 276, 1750-1761.  
18156340 Z.Xiao, H.Bergeron, S.Grosse, M.Beauchemin, M.L.Garron, D.Shaya, T.Sulea, M.Cygler, and P.C.Lau (2008).
Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature-guided sequence alignment.
  Appl Environ Microbiol, 74, 1183-1189.
PDB codes: 2qx3 2qxz 2qy1
17203387 A.F.Mehl, B.Demeler, and A.Zraikat (2007).
A water mediated electrostatic interaction gives thermal stability to the "tail" region of the GrpE protein from E. coli.
  Protein J, 26, 239-245.  
16541263 N.Zheng, B.Huang, J.Xu, S.Huang, J.Chen, X.Hu, K.Ying, and X.Yu (2006).
Enzymatic and physico-chemical characteristics of recombinant cMDH and mMDH of Clonorchis sinensis.
  Parasitol Res, 99, 174-180.  
15857780 V.G.Eijsink, S.Gåseidnes, T.V.Borchert, and B.van den Burg (2005).
Directed evolution of enzyme stability.
  Biomol Eng, 22, 21-30.  
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