Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher...

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Bibliographic Details
Published in:Protein engineering 1998-10, Vol.11 (10), p.925-930
Main Authors: Fiorentino, G, Cannio, R, Rossi, M, Bartolucci, S
Format: Article
Language:English
Subjects:
Alcohol Dehydrogenase - chemistry
Alcohol Dehydrogenase - genetics
Alcohol Dehydrogenase - isolation & purification
Alcohol Dehydrogenase - metabolism
Alcohols - metabolism
Amino Acid Substitution
Bacillus stearothermophilus
Binding Sites
Catalytic Domain - genetics
Enzyme Stability
Escherichia coli - genetics
Geobacillus stearothermophilus - enzymology
Glutamic Acid - genetics
Glutamic Acid - metabolism
Guanidine
Hydrogen-Ion Concentration
Kinetics
Mutagenesis, Site-Directed
NAD - metabolism
Proline - genetics
Proline - metabolism
Protein Denaturation
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Substrate Specificity
Temperature
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Summary:The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects.
ISSN:0269-2139
1741-0126
1741-0134
DOI:10.1093/protein/11.10.925