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 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.
Fiorentino
G
G
Dipartimento di Chimica Organica e Biologica, Università degli Studi di Napoli Federico II, Naples, Italy.
Cannio
R
R
Rossi
M
M
Bartolucci
S
S
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000438:Alcohols; D011994:Recombinant Proteins; D009243:NAD; D018698:Glutamic Acid; D011392:Proline; D000426:Alcohol Dehydrogenase; D019791:Guanidine