Protein Variants | Comment | Organism |
---|---|---|
C295A | the mutant shows an increased size of the alkyl group which can bind in the substrate small pocket by one carbon atom compared to the wild-type enzyme | Thermoanaerobacter ethanolicus |
I86A | the mutant shows altered substrate specificity and expands substrate specificity to include acetophenone, which is a very poor substrate for wild-type SADH, but also reverses the usual preferred stereochemistry to produce the anti-prelog R-product. I86A SADH exhibits limited reactivity with substituted acetophenones, fluorine being the only tolerable substituent | Thermoanaerobacter ethanolicus |
I86A/C295A | the mutant enzyme shows broadened substrate specificity for aryl ketones and broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones compared to the wild-type enzyme. The increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site | Thermoanaerobacter ethanolicus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Dichloromethane | - |
Thermoanaerobacter ethanolicus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Thermoanaerobacter ethanolicus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | required | Thermoanaerobacter ethanolicus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermoanaerobacter ethanolicus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
native enzyme by a procedure including RedA-agarose affinity chromatography to electrophoretical homogeneity | Thermoanaerobacter ethanolicus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
1-(2-pyridyl)acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(2-pyridyl)ethanol + NADP+ | - |
r | |
1-(2-thienyl)acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(2-thienyl)ethanol + NADP+ | - |
r | |
1-(3-pyridyl)acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3-pyridyl)ethanol + NADP+ | - |
r | |
1-(3-thienyl)acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3-thienyl)ethanol + NADP+ | - |
r | |
1-(4-pyridyl)acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(4-pyridyl)ethanol + NADP+ | - |
r | |
1-phenylpropanone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-phenylpropanol + NADP+ | - |
r | |
2',4'-difluoro-acetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(2',4'-difluoro)-phenylethanol + NADP+ | - |
r | |
2'-methylacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(2'-methyl)phenylethanol + NADP+ | - |
r | |
3'-bromo-acetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3'-bromo-)phenylethanol + NADP+ | - |
r | |
3'-chloroacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3'-chlorophenyl)ethanol + NADP+ | - |
r | |
3'-iodoacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3'-iodophenyl)ethanol + NADP+ | - |
r | |
3'-methoxyacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3'-methoxyphenyl)ethanol + NADP+ | - |
r | |
3'-methylacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-(3'-methylphenyl)ethanol + NADP+ | - |
r | |
3'-trifluoromethyl-acetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-[(3'-trifluoromethyl)phenyl]ethanol + NADP+ | - |
r | |
3'-trifluoromethylacetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-[(3'-trifluoromethyl)phenyl]ethanol + NADP+ | - |
r | |
acetone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 2-propanol + NADP+ | - |
r | |
acetophenone + NADPH + H+ | - |
Thermoanaerobacter ethanolicus | 1-phenylethanol + NADP+ | - |
r | |
additional information | the enzyme mutant I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates, it shows broad substrate specificity for meta-substituted, but not para-substituted, acetophenones and aryl ketones | Thermoanaerobacter ethanolicus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 37700, SDS-PAGE | Thermoanaerobacter ethanolicus |
Synonyms | Comment | Organism |
---|---|---|
SADH | - |
Thermoanaerobacter ethanolicus |
secondary alcohol dehydrogenase | - |
Thermoanaerobacter ethanolicus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
- |
Thermoanaerobacter ethanolicus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.04 | - |
3'-trifluoromethyl-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.08 | - |
3'-methoxy-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.18 | - |
2'-methylacetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.47 | - |
1-(3-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
0.61 | - |
3'-iodo-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.66 | - |
3'-chloro-acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.01 | - |
acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.5 | - |
1-(2-thienyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
1.7 | - |
1-(4-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.7 | - |
1-(2-thienyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
2 | - |
3'-methylacetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
2.2 | - |
2',4'-difluoro-acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
2.31 | - |
1-(2-pyridyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
2.9 | - |
2',4'-difluoro-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
6.5 | - |
1-phenylpropanone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
6.5 | - |
3'-bromo-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
6.9 | - |
1-(2-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
8.2 | - |
acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
12.3 | - |
3'-chloro-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
13.1 | - |
1-(4-pyridyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
14.9 | - |
1-(3-thienyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
15.8 | - |
acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
23.3 | - |
1-phenylpropanone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
35 | - |
acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
- |
Thermoanaerobacter ethanolicus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Thermoanaerobacter ethanolicus | |
NADPH | - |
Thermoanaerobacter ethanolicus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.018 | - |
2'-methylacetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.025 | - |
3'-trifluoromethyl-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.16 | - |
3'-iodo-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.18 | - |
3'-methylacetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.2 | - |
1-(3-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
0.21 | - |
3'-methoxy-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.23 | - |
2',4'-difluoro-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.26 | - |
1-(2-pyridyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.26 | - |
1-(2-thienyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.35 | - |
1-(2-thienyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.41 | - |
3'-chloro-acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
0.51 | - |
1-(2-thienyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
0.62 | - |
2',4'-difluoro-acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
0.63 | - |
1-(3-pyridyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
0.65 | - |
3'-bromo-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
1.05 | - |
1-(4-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.08 | - |
acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
1.3 | - |
3'-chloro-acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
1.33 | - |
acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.5 | - |
1-phenylpropanone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
1.6 | - |
1-(2-pyridyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
1.9 | - |
acetophenone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
2.4 | - |
1-(4-pyridyl)acetone | pH 6.5, 50°C, wild-type enzyme | Thermoanaerobacter ethanolicus | |
4.3 | - |
1-(3-thienyl)acetone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
8.1 | - |
1-phenylpropanone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus | |
14.9 | - |
acetophenone | pH 6.5, 50°C, mutant I86A | Thermoanaerobacter ethanolicus |