1JQB

Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability

Summary for 1JQB

• Entry DOI: 10.2210/pdb1jqb/pdb
• Related: 1KEV
• Descriptor: NADP-dependent Alcohol Dehydrogenase, ZINC ION (3 entities in total)
• Functional Keywords: tetramer of 222 symmetry, water-mediated intersubunit salt bridges, rossmann fold, oxidoreductase
• Biological source: Clostridium beijerinckii
• Total number of polymer chains: 4
• Total formula weight: 151709.48

Authors

Levin, I.,Frolow, F.,Bogin, O.,Peretz, M.,Hacham, Y.,Burstein, Y. (deposition date: 2001-08-05, release date: 2002-11-13, Last modification date: 2023-08-16)

Primary citation

Bogin, O.,Levin, I.,Hacham, Y.,Tel-Or, S.,Peretz, M.,Frolow, F.,Burstein, Y.
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, 2002

PubMed 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.

PubMed: 12381840
DOI: 10.1110/ps.0222102

Experimental method

X-RAY DIFFRACTION (1.97 Å)