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PDBsum entry 2tmg

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protein Protein-protein interface(s) links
Oxidoreductase PDB id
2tmg
Jmol
Contents
Protein chains
(+ 0 more) 408 a.a. *
* Residue conservation analysis
PDB id:
2tmg
Name: Oxidoreductase
Title: Thermotoga maritima glutamate dehydrogenase mutant s128r, t158e, n117r, s160e
Structure: Protein (glutamate dehydrogenase). Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes
Source: Thermotoga maritima. Organism_taxid: 2336. Cellular_location: cytoplasm. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Hexamer (from PQS)
Resolution:
2.90Å     R-factor:   0.211     R-free:   0.274
Authors: S.Knapp,J.H.G.Lebbink,J.Van Der Oost,W.M.De Vos,D.Rice, R.Ladenstein
Key ref:
J.H.Lebbink et al. (1999). Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface. J Mol Biol, 289, 357-369. PubMed id: 10366510 DOI: 10.1006/jmbi.1999.2779
Date:
04-Dec-98     Release date:   08-Dec-99    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P96110  (DHE3_THEMA) -  Glutamate dehydrogenase
Seq:
Struc:
416 a.a.
408 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.1.4.1.3  - Glutamate dehydrogenase (NAD(P)(+)).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: L-glutamate + H2O + NAD(P)(+) = 2-oxoglutarate + NH3 + NAD(P)H
L-glutamate
+ H(2)O
+ NAD(P)(+)
= 2-oxoglutarate
+ NH(3)
+ NAD(P)H
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   2 terms 
  Biochemical function     oxidoreductase activity     3 terms  

 

 
    reference    
 
 
DOI no: 10.1006/jmbi.1999.2779 J Mol Biol 289:357-369 (1999)
PubMed id: 10366510  
 
 
Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.
J.H.Lebbink, S.Knapp, J.van der Oost, D.Rice, R.Ladenstein, W.M.de Vos.
 
  ABSTRACT  
 
The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. Schematic presentation of the 18-residue ion-pair network in P. furiosus GDH (a) and the homologous residues in T. maritima GDH (b). Residues forming ion-pairs are connected by dotted lines, broken lines indicate subunit interfaces.
Figure 4.
Figure 4. Stereoview of the 16-residue network in T. maritima quadruple mutant GDH.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 289, 357-369) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
17692336 A.T.Clark, K.Smith, R.Muhandiram, S.P.Edmondson, and J.W.Shriver (2007).
Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d.
  J Mol Biol, 372, 992.  
16759231 M.Karlström, I.H.Steen, D.Madern, A.E.Fedöy, N.K.Birkeland, and R.Ladenstein (2006).
The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima.
  FEBS J, 273, 2851-2868.
PDB code: 1zor
16533850 S.Melchionna, R.Sinibaldi, and G.Briganti (2006).
Explanation of the stability of thermophilic proteins based on unique micromorphology.
  Biophys J, 90, 4204-4212.  
15326599 B.N.Dominy, H.Minoux, and C.L.Brooks (2004).
An electrostatic basis for the stability of thermophilic proteins.
  Proteins, 57, 128-141.  
12824494 K.B.Wong, C.F.Lee, S.H.Chan, T.Y.Leung, Y.W.Chen, and M.Bycroft (2003).
Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer.
  Protein Sci, 12, 1483-1495.
PDB codes: 1go0 1go1
12012341 B.Cobucci-Ponzano, M.Moracci, B.Di Lauro, M.Ciaramella, R.D'Avino, and M.Rossi (2002).
Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization.
  Proteins, 48, 98.  
12382287 C.Charron, B.Vitoux, and A.Aubry (2002).
Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus.
  Biopolymers, 65, 263-273.  
17590951 D.Perl, and F.X.Schmid (2002).
Some like it hot: the molecular determinants of protein thermostability.
  Chembiochem, 3, 39-44.  
12202384 S.Kumar, and R.Nussinov (2002).
Relationship between ion pair geometries and electrostatic strengths in proteins.
  Biophys J, 83, 1595-1612.  
11551792 A.Karshikoff, and R.Ladenstein (2001).
Ion pairs and the thermotolerance of proteins from hyperthermophiles: a "traffic rule" for hot roads.
  Trends Biochem Sci, 26, 550-556.  
11453986 B.Clantin, C.Tricot, T.Lonhienne, V.Stalon, and V.Villeret (2001).
Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.
  Eur J Biochem, 268, 3937-3942.  
11391775 C.A.Olson, E.J.Spek, Z.Shi, A.Vologodskii, and N.R.Kallenbach (2001).
Cooperative helix stabilization by complex Arg-Glu salt bridges.
  Proteins, 44, 123-132.  
11238984 C.Vieille, and G.J.Zeikus (2001).
Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.
  Microbiol Mol Biol Rev, 65, 1.  
11258921 M.Nakasako, T.Fujisawa, S.Adachi, T.Kudo, and S.Higuchi (2001).
Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering.
  Biochemistry, 40, 3069-3079.
PDB code: 1euz
10651277 C.Li, J.Heatwole, S.Soelaiman, and M.Shoham (1999).
Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability.
  Proteins, 37, 619-627.
PDB code: 1bxz
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.