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Literature summary for 1.1.1.B4 extracted from

  • Nealon, C.M.; Welsh, T.P.; Kim, C.S.; Phillips, R.S.
    I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones (2016), Arch. Biochem. Biophys., 606, 151-156 .
    View publication on PubMed
Search for more literature for 1.1.1.B4

Protein Variants

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

Inhibitors Comment Organism Structure
Dichloromethane
-
 Thermoanaerobacter ethanolicus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Thermoanaerobacter ethanolicus

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+ required Thermoanaerobacter ethanolicus

Organism

Organism UniProt Comment Textmining
Thermoanaerobacter ethanolicus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
native enzyme by a procedure including RedA-agarose affinity chromatography to electrophoretical homogeneity Thermoanaerobacter ethanolicus

Substrates and Products (Substrate)

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

Subunits Comment Organism
? x * 37700, SDS-PAGE Thermoanaerobacter ethanolicus

Synonyms

Synonyms Comment Organism
SADH
-
 Thermoanaerobacter ethanolicus
secondary alcohol dehydrogenase
-
 Thermoanaerobacter ethanolicus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
 Thermoanaerobacter ethanolicus

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
-
 Thermoanaerobacter ethanolicus

Cofactor

Cofactor Comment Organism Structure
NADP+
-
 Thermoanaerobacter ethanolicus
NADPH
-
 Thermoanaerobacter ethanolicus

kcat/KM [mM/s]

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