1jqb
From Proteopedia
(New page: 200px<br /><applet load="1jqb" size="450" color="white" frame="true" align="right" spinBox="true" caption="1jqb, resolution 1.97Å" /> '''Alcohol Dehydrogenas...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1jqb.gif|left|200px]]<br /><applet load="1jqb" size=" | + | [[Image:1jqb.gif|left|200px]]<br /><applet load="1jqb" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1jqb, resolution 1.97Å" /> | caption="1jqb, resolution 1.97Å" /> | ||
'''Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability'''<br /> | '''Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Previous research in our laboratory comparing the three-dimensional | + | 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 Structure== | ==About this Structure== | ||
| - | 1JQB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Clostridium_beijerinckii Clostridium beijerinckii] with ZN as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [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] Full crystallographic information is available from [http:// | + | 1JQB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Clostridium_beijerinckii Clostridium beijerinckii] with <scene name='pdbligand=ZN:'>ZN</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [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] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JQB OCA]. |
==Reference== | ==Reference== | ||
| Line 25: | Line 25: | ||
[[Category: water-mediated intersubunit salt bridges]] | [[Category: water-mediated intersubunit salt bridges]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:25:34 2008'' |
Revision as of 11:25, 21 February 2008
|
Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability
Overview
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 Structure
1JQB is a Single protein structure of sequence from Clostridium beijerinckii with as ligand. Active as Alcohol dehydrogenase (NADP(+)), with EC number 1.1.1.2 Full crystallographic information is available from OCA.
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:12381840
Page seeded by OCA on Thu Feb 21 13:25:34 2008
