Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.


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. Study holds ProTherm entries: 17554, 17555, 17556, 17557, 17558, 17559 Extra Details: Alcohol dehydrogenase; Clostridium beijerinckii; Thermoanaerobacter brockii; site-directed mutagenesis; ion-pair network; salt bridge; thermostability

Submission Details

ID: FMC2sdMt

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Bogin O;Levin I;Hacham Y;Tel-Or S;Peretz M;Frolow F;Burstein Y,Protein Sci. (2002) Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging. PMID:12381840
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1JQB 2001-08-05T00:00:00+0000 1.97 Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability
1KEV 1996-10-21T00:00:00+0000 2.05 STRUCTURE OF NADP-DEPENDENT ALCOHOL DEHYDROGENASE
1PED 1995-12-28T00:00:00+0000 2.15 BACTERIAL SECONDARY ALCOHOL DEHYDROGENASE (APO-FORM)
2B83 2005-10-06T00:00:00+0000 2.25 A single amino acid substitution in the Clostridium beijerinckii alcohol dehydrogenase is critical for thermostabilization
3FPL 2009-01-05T00:00:00+0000 1.9 Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of C. beijerinckii ADH by T. brockii ADH
3FSR 2009-01-11T00:00:00+0000 2.2 Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of T. brockii ADH by C. beijerinckii ADH
3FTN 2009-01-13T00:00:00+0000 2.19 Q165E/S254K Double Mutant Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of T. brockii ADH by C. beijerinckii ADH
6SCH 2019-07-24T00:00:00+0000 2.2 NADH-dependent variant of CBADH

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 NADP-dependent isopropanol dehydrogenase P25984 ADH_CLOBE