Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.


Abstract

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability. Study holds ProTherm entries: 8918, 8919, 8920, 8921, 8922, 8923, 8924, 8925, 8926, 8927, 8928, 8929, 8930 Extra Details: CBADH alcohol dehydrogenase; clostridium beijerinckii; entamoeba histolytica;,mutagenesis; proline; thermoanaerobacter brockii; thermostability

Submission Details

ID: JwDvvaWB3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Bogin O;Peretz M;Hacham Y;Korkhin Y;Frolow F;Kalb(Gilboa) AJ;Burstein Y,Protein Sci. (1998) Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase. PMID:9836874
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
3FPL 2010-01-19 1.9 Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of C. beijerinckii ADH by T. brockii ADH
1JQB 2002-11-13 1.97 Alcohol Dehydrogenase from Clostridium Beijerinckii: Crystal Structure of Mutant with Enhanced Thermal Stability
1KEV 1997-10-22 2.05 STRUCTURE OF NADP-DEPENDENT ALCOHOL DEHYDROGENASE
1PED 1997-07-07 2.15 BACTERIAL SECONDARY ALCOHOL DEHYDROGENASE (APO-FORM)
3FTN 2010-01-26 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
3FSR 2010-01-19 2.2 Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of T. brockii ADH by C. beijerinckii ADH
2B83 2006-09-19 2.25 A single amino acid substitution in the Clostridium beijerinckii alcohol dehydrogenase is critical for thermostabilization

Relevant UniProtKB Entries

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