1jqb: Difference between revisions

Revision as of 21:37, 2 May 2008

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

OverviewOverview

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.

About this StructureAbout this Structure

1JQB is a Single protein structure of sequence from Clostridium beijerinckii. Full crystallographic information is available from OCA.

ReferenceReference

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging., Bogin O, Levin I, Hacham Y, Tel-Or S, Peretz M, Frolow F, Burstein Y, Protein Sci. 2002 Nov;11(11):2561-74. PMID:12381840 Page seeded by OCA on Fri May 2 21:37:56 2008

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OCA

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 [[Image:1jqb.gif|left|200px]]
- {{Structure
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-|ACTIVITY= <span class='plainlinks'>[http://en.wikipedia.org/wiki/Alcohol_dehydrogenase_(NADP(+)) Alcohol dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.2 1.1.1.2] </span>
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-}}
 '''Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability'''
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 ==Reference==
 Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging., Bogin O, Levin I, Hacham Y, Tel-Or S, Peretz M, Frolow F, Burstein Y, Protein Sci. 2002 Nov;11(11):2561-74. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/12381840 12381840]
-[[Category: Alcohol dehydrogenase (NADP(+))]]
 [[Category: Clostridium beijerinckii]]
 [[Category: Single protein]]
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 [[Category: Levin, I.]]
 [[Category: Peretz, M.]]
-[[Category: rossmann fold]]
+[[Category: Rossmann fold]]
-[[Category: tetramer of 222 symmetry]]
+[[Category: Tetramer of 222 symmetry]]
-[[Category: water-mediated intersubunit salt bridge]]
+[[Category: Water-mediated intersubunit salt bridge]]
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May  2 21:37:56 2008''
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 21:37:23 2008''