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PDBsum entry 1jqb
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protein metals Protein-protein interface(s) links
Oxidoreductase PDB id
1jqb

 

 

 

 

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Contents
Protein chains
351 a.a. *
Metals
_ZN ×4
Waters ×411
* Residue conservation analysis
PDB id:
1jqb
Name: Oxidoreductase
Title: Alcohol dehydrogenase from clostridium beijerinckii: crystal structure of mutant with enhanced thermal stability
Structure: NADP-dependent alcohol dehydrogenase. Chain: a, b, c, d. Engineered: yes. Mutation: yes
Source: Clostridium beijerinckii. Organism_taxid: 1520. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
Resolution:
1.97Å     R-factor:   0.219     R-free:   0.247
Authors: I.Levin,F.Frolow,O.Bogin,M.Peretz,Y.Hacham,Y.Burstein
Key ref:
O.Bogin et al. (2002). Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging. Protein Sci, 11, 2561-2574. PubMed id: 12381840 DOI: 10.1110/ps.0222102
Date:
05-Aug-01     Release date:   13-Nov-02    
PROCHECK
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 Headers
 References

Protein chains
Pfam   ArchSchema ?
P25984  (ADH_CLOBE) -  NADP-dependent isopropanol dehydrogenase from Clostridium beijerinckii
Seq:
Struc: 		351 a.a.
351 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 4 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.1.1.1.80  - isopropanol dehydrogenase (NADP(+)).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: propan-2-ol + NADP+ = acetone + NADPH + H+
propan-2-ol
 + NADP(+)
 = acetone
 + NADPH
 + H(+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 

 
    reference    
 
 
DOI no: 10.1110/ps.0222102 Protein Sci 11:2561-2574 (2002)
PubMed id: 12381840  
 
 
Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.
O.Bogin, I.Levin, Y.Hacham, S.Tel-Or, M.Peretz, F.Frolow, Y.Burstein.
 
  ABSTRACT  
 
Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.
 
  Selected figure(s)  
 
Figure 3.
Fig. 3. Three-dimensional structure of the tetramer of the quadruple mutant of CbADH. The four subunits are: A (yellow-light green), B (red), C (blue), and D (dark green). (A) Stereoview of the hydrogen-bond network at the interface of three subunits of the quadruple mutant of CbADH. Residues Asp237, Lys257, Arg238, Ser108, and Glu165 from Subunit A, Arg304 from Subunit B, and Ala97 of subunit D (via its main-chain carbonyl group) participate in this network. The water molecules are shown in magenta, hydrogen bond in white dashed lines, and distances are in Å. '2Fo-Fc' electron density map of the quadruple mutant is contoured at 1.7 level. (B) Extra salt bridges (magenta) in subunit A (the yellow coil) in the Q165E/M304R mutant. 		Figure 5.
Fig. 5. Two alternative conformations of Lys254 of the quadruple mutant of CbADH. (A) In subunit A, the -amino group of Lys254 points towards Glu280 to form a salt bridge; however neither hydrogen-bond connection nor electrostatic interaction between Lys254 and Glu224 is observed. (B) In subunit D, Lys254 is equally distanced from both Glu280 and Glu224; it interacts electrostatically with both of them and forms an additional hydrogen bond via a water molecule with Glu224. '2Fo-Fc' electron density map is contoured at 1.2 .
 
  The above figures are reprinted by permission from the Protein Society: Protein Sci (2002, 11, 2561-2574) copyright 2002.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
18780158 A.K.Williamson (2008).
Structural and functional aspects of the MSP (PsbO) and study of its differences in thermophilic versus mesophilic organisms.
  Photosynth Res, 98, 365-389.  
18260103 E.Goihberg, O.Dym, S.Tel-Or, L.Shimon, F.Frolow, M.Peretz, and Y.Burstein (2008).
Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution.
  Proteins, 72, 711-719.
PDB codes: 2nvb 2oui
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.  
17063493 E.Goihberg, O.Dym, S.Tel-Or, I.Levin, M.Peretz, and Y.Burstein (2007).
A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase.
  Proteins, 66, 196-204.
PDB code: 2b83
17351092 W.Zhang, E.J.Mullaney, and X.G.Lei (2007).
Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase.
  Appl Environ Microbiol, 73, 3069-3076.  
16084384 R.A.Frank, J.V.Pratap, X.Y.Pei, R.N.Perham, and B.F.Luisi (2005).
The molecular origins of specificity in the assembly of a multienzyme complex.
  Structure, 13, 1119-1130.
PDB code: 2bp7
15858262 Y.Eisenberg-Domovich, V.P.Hytönen, M.Wilchek, E.A.Bayer, M.S.Kulomaa, and O.Livnah (2005).
High-resolution crystal structure of an avidin-related protein: insight into high-affinity biotin binding and protein stability.
  Acta Crystallogr D Biol Crystallogr, 61, 528-538.
PDB codes: 1y52 1y53 1y55
14997561 A.W.Partridge, A.G.Therien, and C.M.Deber (2004).
Missense mutations in transmembrane domains of proteins: phenotypic propensity of polar residues for human disease.
  Proteins, 54, 648-656.  
12943843 J.K.Yano, and T.L.Poulos (2003).
New understandings of thermostable and peizostable enzymes.
  Curr Opin Biotechnol, 14, 360-365.  
12837777 Z.Xu, Y.Liu, Y.Yang, W.Jiang, E.Arnold, and J.Ding (2003).
Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability.
  J Bacteriol, 185, 4038-4049.
PDB code: 1nfg
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 codes are shown on the right.

 

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