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Literature summary for 1.1.1.1 extracted from

  • Campbell, E.; Wheeldon, I.; Banta, S.
    Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior (2010), Biotechnol. Bioeng., 107, 763-774 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli Pyrococcus furiosus

Protein Variants

Protein Variants Comment Organism
H255R the H255R single mutant exhibits an increased binding affinity toward NADP+ and a concomitant reduction in affinity for NAD+. The apparent kcat for H255R is about 60% of that of the wild-type with NAD+, but it is sixfold higher than the wild-type with NADP+. Position 255 is important for recognizing NADP(H), but is not the sole determinant of cofactor specificity Pyrococcus furiosus
K249G catalytic efficiency increases 5fold for NADH, the efficiency with NADPH increases more than 30fold in comparison to the wild-type Pyrococcus furiosus
K249G/H255R the catalytic efficiency with NADH increases 4fold, the efficiency with NADPH increases more than 16fold. In the oxidation reaction, the kcat with NAD+ improves by 15fold for the double mutant over the wild type enzyme. With NADP+ the kcat is nearly two orders of magnitude larger than the wild-type. Mutant exhibits significantly improved activity and broadened cofactor specificity as compared to the wild-type enzyme Pyrococcus furiosus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0051
-
NADP+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, wild-type enzyme Pyrococcus furiosus
0.033
-
NADPH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, mutant enzyme K249G/H255R Pyrococcus furiosus
0.05
-
NADH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, mutant enzyme K249G/H255R Pyrococcus furiosus
0.063
-
NAD+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, wild-type enzyme Pyrococcus furiosus
0.078
-
NADP+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, mutant enzyme K249G/H255R Pyrococcus furiosus
0.19
-
NADH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, wild-type enzyme Pyrococcus furiosus
0.28
-
NADPH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, wild-type enzyme Pyrococcus furiosus
0.46
-
NAD+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, mutant enzyme K249G/H255R Pyrococcus furiosus
0.9
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADH, wild-type enzyme Pyrococcus furiosus
1.3
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NADP+, wild-type enzyme Pyrococcus furiosus
5
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADPH, mutant enzyme K249G/H255R Pyrococcus furiosus
6.7
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADPH, wild-type enzyme Pyrococcus furiosus
13
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADH, mutant enzyme K249G/H255R Pyrococcus furiosus
29
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NAD+, wild-type enzyme Pyrococcus furiosus
200
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NADP+, mutant enzyme K249G/H255R Pyrococcus furiosus
690
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NAD+, mutant enzyme K249G/H255R Pyrococcus furiosus

Organism

Organism UniProt Comment Textmining
Pyrococcus furiosus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Pyrococcus furiosus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,3-butanediol + NAD+
-
Pyrococcus furiosus 3-hydroxy-2-butanone + NADH + H+
-
r
2,3-butanediol + NADP+
-
Pyrococcus furiosus 3-hydroxy-2-butanone + NADPH + H+
-
?
3-hydroxy-2-butanone + NADH + H+
-
Pyrococcus furiosus 2,3-butanediol + NAD+
-
r
3-hydroxy-2-butanone + NADPH + H+
-
Pyrococcus furiosus 2,3-butanediol + NADP+
-
r

Synonyms

Synonyms Comment Organism
AdhD
-
Pyrococcus furiosus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
assay at Pyrococcus furiosus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.03
-
NADP+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, wild-type enzyme Pyrococcus furiosus
0.03
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NADP+, wild-type enzyme Pyrococcus furiosus
0.2
-
NADPH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, wild-type enzyme Pyrococcus furiosus
0.2
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADPH, wild-type enzyme Pyrococcus furiosus
0.36
-
NADH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, wild-type enzyme Pyrococcus furiosus
0.36
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADH, wild-type enzyme Pyrococcus furiosus
1
-
NAD+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, wild-type enzyme Pyrococcus furiosus
1
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NAD+, wild-type enzyme Pyrococcus furiosus
1.1
-
NADH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, mutant enzyme K249G/H255R Pyrococcus furiosus
1.1
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADH, mutant enzyme K249G/H255R Pyrococcus furiosus
1.2
-
NADPH 45°C, pH 6.1, cosubstrate: 3-hydroxy-2-butanone, mutant enzyme K249G/H255R Pyrococcus furiosus
1.2
-
3-hydroxy-2-butanone 45°C, pH 6.1, cosubstrate: NADPH, mutant enzyme K249G/H255R Pyrococcus furiosus
4.7
-
NADP+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, mutant enzyme K249G/H255R Pyrococcus furiosus
4.7
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NADP+, mutant enzyme K249G/H255R Pyrococcus furiosus
15
-
NAD+ 45°C, pH 8.8, cosubstrate: 2,3-butanediol, mutant enzyme K249G/H255R Pyrococcus furiosus
15
-
2,3-Butanediol 45°C, pH 8.8, cosubstrate: NAD+, mutant enzyme K249G/H255R Pyrococcus furiosus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1
-
assay at, reduction of 3-hydroxy-2-butanone Pyrococcus furiosus
8.8
-
assay at, oxidation of 2,3-butanediol Pyrococcus furiosus

Cofactor

Cofactor Comment Organism Structure
NAD+ H255R single mutant exhibits an increased binding affinity toward NADP+ and a concomitant reduction in affinity for NAD+ Pyrococcus furiosus
NADH
-
Pyrococcus furiosus
NADP+ H255R single mutant exhibits an increased binding affinity toward NADP+ and a concomitant reduction in affinity for NAD+ Pyrococcus furiosus
NADPH experiments with the highly active double mutant K249G/H255R using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration Pyrococcus furiosus