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Literature summary for 1.1.1.86 extracted from

  • Tyagi, R.; Lee, Y.T.; Guddat, L.W.; Duggleby, R.G.
    Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site (2005), FEBS J., 272, 593-602.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
-
Escherichia coli

Protein Variants

Protein Variants Comment Organism
D217E less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
D217N less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E213D 75% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
E213Q less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
E221D less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E221Q less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E389D less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E389Q less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E393D less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
E393Q the mutant is insoluble, a soluble form is obtained only after denaturation Escherichia coli
H132K less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
H132Q less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
K155E less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
K155Q less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
K155R less than 4% reductoisomerase activity in comparison of wild-type enzyme Escherichia coli
S414A less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli
S414T less than 4% reductoisomerase activity in comparison to wild-type enzyme Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00016
-
NADPH pH 8.0, 37°C, mutant E213D Escherichia coli
0.00253
-
NADPH pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.0031
-
NADPH pH 8.0, 37°C, mutant H132K Escherichia coli
0.0048
-
NADPH pH 8.0, 37°C, mutant E393D Escherichia coli
0.005
-
NADPH pH 8.0, 37°C, mutant D217N Escherichia coli
0.005
-
NADPH pH 8.0, 37°C, mutant S414T Escherichia coli
0.0073
-
NADPH pH 8.0, 37°C, mutant K155R Escherichia coli
0.00804
-
NADPH pH 8.0, 37°C, mutant K155E Escherichia coli
0.0084
-
NADPH pH 8.0, 37°C, mutant S414A Escherichia coli
0.0093
-
NADPH pH 8.0, 37°C, mutant K155Q Escherichia coli
0.02
-
NADPH pH 8.0, 37°C, mutant E221D Escherichia coli
0.02
-
NADPH pH 8.0, 37°C, mutant E221Q Escherichia coli
0.023
-
NADPH pH 8.0, 37°C, mutant E389D Escherichia coli
0.069
-
NADPH pH 8.0, 37°C, mutant H132Q Escherichia coli
0.08
-
NADPH pH 8.0, 37°C, mutant D217E Escherichia coli
0.17
-
2-ketopantoate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.21
-
3-hydroxy-2-ketobutyrate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.25
-
2-acetolactate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.27
-
3-hydroxy-3-methyl-2-ketobutyrate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.334
-
3-hydroxypyruvate pH 8.0, 37°C, mutant S414A Escherichia coli
0.356
-
2-acetolactate pH 8.0, 37°C, mutant E221D Escherichia coli
0.414
-
2-acetolactate pH 8.0, 37°C, mutant S414T Escherichia coli
0.441
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E213Q Escherichia coli
0.588
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E393Q Escherichia coli
0.711
-
2-acetolactate pH 8.0, 37°C, mutant S414A Escherichia coli
0.818
-
3-hydroxypyruvate pH 8.0, 37°C, mutant H132K Escherichia coli
0.922
-
2-acetolactate pH 8.0, 37°C, mutant H213D Escherichia coli
0.929
-
2-acetolactate pH 8.0, 37°C, mutant H132Q Escherichia coli
1.101
-
3-hydroxypyruvate pH 8.0, 37°C, mutant S414T Escherichia coli
1.218
-
2-acetolactate pH 8.0, 37°C, mutant H155R Escherichia coli
1.37
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E221D Escherichia coli
1.54
-
pyruvate pH 8.0, 37°C, wild-type enzyme Escherichia coli
2.028
-
2-acetolactate pH 8.0, 37°C, mutant E289D Escherichia coli
2.66
-
3-hydroxypyruvate pH 8.0, 37°C, mutant K155E Escherichia coli
2.96
-
3-hydroxypyruvate pH 8.0, 37°C, wild-type enzyme Escherichia coli
3.15
-
2-ketovalerate pH 8.0, 37°C, wild-type enzyme Escherichia coli
3.32
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E393D Escherichia coli
3.67
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E213D Escherichia coli
4.56
-
2-Ketobutyrate pH 8.0, 37°C, wild-type enzyme Escherichia coli
6.91
-
2-ketoisovalerate pH 8.0, 37°C, wild-type enzyme Escherichia coli
7.43
-
3-hydroxypyruvate pH 8.0, 37°C, mutant H132Q Escherichia coli
7.64
-
3-hydroxypyruvate pH 8.0, 37°C, mutant D217N Escherichia coli
8.5
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E389D Escherichia coli
8.88
-
3-hydroxypyruvate pH 8.0, 37°C, mutant E389Q Escherichia coli
13.6
-
3-hydroxypyruvate pH 8.0, 37°C, mutant K155R Escherichia coli
15.3
-
3-hydroxypyruvate pH 8.0, 37°C, mutant K155Q Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ the reaction requires a divalent metal ion Escherichia coli
Mg2+ the reaction requires a divalent metal ion Escherichia coli
Mn2+ the reaction requires a divalent metal ion Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Escherichia coli the enzyme is involved in the biosynthesis of the branched-chain amino acids ?
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
of wild-type and mutants Escherichia coli

Reaction

Reaction Comment Organism Reaction ID
(2R)-2,3-dihydroxy-3-methylbutanoate + NADP+ = (2S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ bifunctional enzyme that catalyses two different reactions at a common active site, an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a-ketoacid Escherichia coli

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
the specific activity with pyruvate is 1% and with 2-ketovalerate, 2-ketopantoate and 2-ketobutyrate is 8% of that of 2-acetolactate, comparison of activities of wild-type and mutant enzymes Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-acetolactate + NADPH + H+
-
Escherichia coli 2,3-dihydroxy-3-methylbutanoate + NADP+
-
r
2-ketobutyrate + NADPH + H+
-
Escherichia coli ?
-
r
2-ketoisovalerate + NADPH + H+
-
Escherichia coli ?
-
r
2-ketopantoate + NADPH + H+
-
Escherichia coli ?
-
r
2-ketovalerate + NADPH + H+
-
Escherichia coli ?
-
r
3-hydroxy-2-ketobutyrate + NADPH + H+
-
Escherichia coli ?
-
r
3-hydroxy-3-methyl-2-ketobutyrate + NADP+
-
Escherichia coli ?
-
r
3-hydroxypyruvate + NADPH + H+
-
Escherichia coli ?
-
r
additional information the enzyme is involved in the biosynthesis of the branched-chain amino acids Escherichia coli ?
-
?
pyruvate + NADPH + H+
-
Escherichia coli ?
-
r

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.021
-
pyruvate pH 8.0, 37°C Escherichia coli
0.05
-
2-ketovalerate pH 8.0, 37°C Escherichia coli
0.167
-
2-Ketobutyrate pH 8.0, 37°C Escherichia coli
0.182
-
2-ketoisovalerate pH 8.0, 37°C Escherichia coli
0.194
-
2-ketopantoate pH 8.0, 37°C Escherichia coli
0.594
-
3-hydroxy-2-ketobutyrate pH 8.0, 37°C Escherichia coli
2.231
-
2-acetolactate pH 8.0, 37°C Escherichia coli
3.511
-
3-hydroxy-3-methyl-2-ketobutyrate pH 8.0, 37°C Escherichia coli
5.376
-
3-hydroxypyruvate pH 8.0, 37°C Escherichia coli

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Escherichia coli
NADPH
-
Escherichia coli