Information on EC 1.1.1.86 - ketol-acid reductoisomerase (NADP+)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.1.1.86
-
RECOMMENDED NAME
GeneOntology No.
ketol-acid reductoisomerase (NADP+)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2R)-2,3-dihydroxy-3-methylbutanoate + NADP+ = (2S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
-
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rearrangement
-
-
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redox reaction
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-
-
-
reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
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isoleucine metabolism
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-
L-isoleucine biosynthesis I (from threonine)
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-
L-isoleucine biosynthesis III
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L-valine biosynthesis
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Metabolic pathways
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Pantothenate and CoA biosynthesis
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pyruvate fermentation to isobutanol (engineered)
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valine metabolism
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Valine, leucine and isoleucine biosynthesis
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-
SYSTEMATIC NAME
IUBMB Comments
(R)-2,3-dihydroxy-3-methylbutanoate:NADP+ oxidoreductase (isomerizing)
Also catalyses the reduction of 2-ethyl-2-hydroxy-3-oxobutanoate to 2,3-dihydroxy-3-methylpentanoate. The enzyme, found in many bacteria and archaea, is specific for NADPH (cf. EC 1.1.1.382, ketol-acid reductoisomerase (NAD+) and EC 1.1.1.383, ketol-acid reductoisomerase [NAD(P)+]).
CAS REGISTRY NUMBER
COMMENTARY hide
9075-02-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
a histatin-resistant derivative of Candida albicans strain 132A is used
-
-
Manually annotated by BRENDA team
Digitaria adscendens
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
morning glory
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene Pi-kari1
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-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
black nightshade
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+
(S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH
show the reaction diagram
-
the enzyme is involved in biosynthesis of the branched chain amino acids valine and leucine, pathway overview
-
-
?
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+
(S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+
show the reaction diagram
2-aceto-2-hydroxybutyrate + NADP+
(2R,3R)-2,3-dihydroxy-3-methylvalerate + NADPH + H+
show the reaction diagram
-
-
-
-
r
2-aceto-2-hydroxybutyrate + NADPH
2,3-dihydroxy-3-methylvalerate + NADP+
show the reaction diagram
2-acetolactate + NADP+
(2R)-2,3-dihydroxy-3-isovalerate + NADPH + H+
show the reaction diagram
-
-
-
-
r
2-acetolactate + NADPH
2,3-dihydroxy-3-methylbutanoate + NADP+
show the reaction diagram
2-acetolactate + NADPH
2,3-dihydroxy-3-methylbutanoate + NADP+ + H+
show the reaction diagram
-
the enzyme is the second of the valine pathway
-
-
r
2-acetolactate + NADPH
2,3-dihydroxyisovalerate + NADP+
show the reaction diagram
2-acetolactate + NADPH + H+
3-hydroxy-2-ketobutyrate + NADP+
show the reaction diagram
-
-
-
-
r
2-ketobutyrate + NADPH + H+
?
show the reaction diagram
-
-
-
-
r
2-ketoisovalerate + NADPH + H+
?
show the reaction diagram
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-
-
-
r
2-ketopantoate + NADPH + H+
?
show the reaction diagram
-
-
-
-
r
2-ketovalerate + NADPH + H+
?
show the reaction diagram
-
-
-
-
r
3-hydroxy-3-methyl-2-ketobutyrate + NADP+
?
show the reaction diagram
-
-
-
-
r
3-hydroxypyruvate + NADPH + H+
?
show the reaction diagram
-
-
-
-
r
acetolactate + NADPH + H+
2,3-dihydroxy-2-methylbutanoate + NADP+
show the reaction diagram
hydroxypyruvate + NADPH
glycerate + NADP+
show the reaction diagram
-
-
-
?
NADP+ + 3-hydroxy-3-methyl-2-oxobutanoate
NADPH + acetolactate
show the reaction diagram
-
-
-
?
NADPH + 2-aceto-2-hydroxybutyrate
NADP+ + ?
show the reaction diagram
NADPH + 2-acetolactate
NADP+ + 3-hydroxy-3-methyl-2-oxobutyrate
show the reaction diagram
NADPH + 2-oxo-3-hydroxyisovalerate
NADP+ + alpha,beta-dihydroxyisovalerate
show the reaction diagram
-
-
-
?
NADPH + alpha-aceto-alpha-hydroxybutyrate
NADP+ + ?
show the reaction diagram
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enzyme of isoleucine biosynthesis
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-
?
pyruvate + NADPH + H+
?
show the reaction diagram
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-
-
r
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+
(S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+
show the reaction diagram
-
-
-
-
r
2-aceto-2-hydroxybutyrate + NADPH
2,3-dihydroxy-3-methylvalerate + NADP+
show the reaction diagram
-
-
-
-
?
2-acetolactate + NADPH
2,3-dihydroxy-3-methylbutanoate + NADP+
show the reaction diagram
2-acetolactate + NADPH
2,3-dihydroxy-3-methylbutanoate + NADP+ + H+
show the reaction diagram
-
the enzyme is the second of the valine pathway
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-
r
2-acetolactate + NADPH
2,3-dihydroxyisovalerate + NADP+
show the reaction diagram
-
-
-
-
?
NADPH + alpha-aceto-alpha-hydroxybutyrate
NADP+ + ?
show the reaction diagram
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enzyme of isoleucine biosynthesis
-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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the reaction requires a divalent metal ion
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,1-cyclopropanedicarboxylic acid
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i.e. CPCA, inhibition in vivo, effect on pea root length, shoot length, and fermentative metabolism, effects of KARI inhibition effects on other enzymes, such as alcohol dehydrogenase, EC 1.1.1.1, pyruvate dehydrogenase, EC 4.1.1.1, lactate dehydrogenase, EC 1.1.1.27, and alanine aminotransferase, EC 2.6.1.2, overview
1-aminocarbonyl-cyclopropane carboxylate
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1-aminocarbonylcyclopropanecarboxylate
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1-carbamoylcyclopropanecarboxylic acid
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1-cyano-cyclopropane carboxylate
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1-cyano-N-(2,4,5-trichlorophenyl)cyclopropanecarboxamide
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inhibition rate: 0%
1-cyano-N-(2,4-dichlorophenyl)cyclopropanecarboxamide
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inhibition rate: 97.04%
1-cyano-N-(2-hydroxyethyl)cyclopropanecarboxamide
-
inhibition rate: 98.92%
1-cyano-N-(2-methylphenyl)cyclopropanecarboxamide
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inhibition rate: 100%
1-cyano-N-(4-methoxyphenyl)cyclopropanecarboxamide
-
inhibition rate: 3.95%
1-cyano-N-(4-methylphenyl)cyclopropanecarboxamide
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inhibition rate: 61.21%
1-cyano-N-phenylcyclopropanecarboxamide
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inhibition rate: 77.23%
1-cyano-N-[(E)-(3,3-dichloroprop-1-yn-1-yl)diazenyl]sulfanylcyclopropanecarboxamide
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inhibition rate: 100%
1-cyano-N-[2-(trifluoromethyl)phenyl]cyclopropanecarboxamide
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inhibition rate: 0%
1-cyano-N-[3-(trifluoromethyl)phenyl]cyclopropanecarboxamide
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inhibition rate: 0%
1-cyano-N-[4-(trifluoromethyl)phenyl]cyclopropanecarboxamide
-
inhibition rate: 0%
1-cyano-N-[[(3-methylcyclopropa-1,2-dien-1-yl)amino]sulfanyl]cyclopropanecarboxamide
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inhibition rate: 69.81%
1-cyanocyclopropanecarboxylate
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1-cyanocyclopropanecarboxylic acid
1-hydroxycyclopropanecarboxylate
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11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11-one
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2,3-dihydroxy-3-isovalerate
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2,3-dihydroxy-3-methylbutanoic acid
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linear noncompetitive inhibitor of both 2-acetolactate and NADPH
2,3-dihydroxy-3-methylbutyrate
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2-(4-benzylpiperazin-1-yl)-N-(3,4-dichlorophenyl)acetamide
2-(4-benzylpiperazin-1-yl)-N-arylacetamide
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-
2-(4-methoxybenzamido)benzoic acid
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2-dimethylphosphinoyl-2-hydroxyacetate
2-dimethylphosphinoyl-2-hydroxyacetic acid
2-hydroxy-2-methyl-3-oxopentanoate
-
-
2-Hydroxy-2-methylbutyrate
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0.01 M 45% inhibition
2-Hydroxybutyrate
-
0.01 M, 48% inhibition
2-Hydroxyisovalerate
2-Methyllactate
2-Oxo-3-hydroxyisovalerate
-
0.001 mM, 58% inhibition
2-oxoisovalerate
-
0.01 M, 21% inhibition
2-[2-(4-morpholino)]acetamido-4-methylthiazole
-
-
2-[[(4-methoxyphenyl)carbonyl]amino]benzoic acid
3-aminopyridine-NADP+
-
-
3-hydroxy-2-oxobutanoic acid
3-hydroxy-3-methyl-2-oxobutanoic acid
4,4'-(pentamethylenedioxy)dibenzamidne bis(2-hydroxyethanesulfonate)
4-(2,4-dichlorophenoxy)benzenecarboximidamide
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4-(2-fluorophenoxy)benzenecarboximidamide
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4-(3-chlorophenoxy)benzenecarboximidamide
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4-(4-chlorophenoxy)benzenecarboximidamide
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4-phenoxybenzenecarboximidamide
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-
arsenite
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slight inhibition
ascorbic acid
-
slight inhibition with 2-acetolactate as substrate
chlorsulfuron
cyclopropane-1,1-dicarboxylate
cyclopropane-1,1-dicarboxylic acid
-
-
dimethylmalonate
-
slow-binding inhibitor
ethyl 1-cyanocyclopropanecarboxylate
-
inhibition rate: 0%
ethyl 3-hydroxy-2-oxobutanoate
ethyl 3-methyl-3-hydroxy-2-oxobutanoate
ethyl [(2-chlorophenyl)(hydroxy)amino](oxo)acetate
-
-
ethyl [hydroxy(2-methylphenyl)amino](oxo)acetate
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-
ethyl [hydroxy(4-methylphenyl)amino](oxo)acetate
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ethyl [[4-(cyanomethyl)phenyl](hydroxy)amino](oxo)acetate
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ethylene glycol
-
exhibits competitive and uncompetitive inhibition
HOE 704
L-ascorbic acid
-
slightly inhibitory with alpha-acetolactate as substrate
methyl [hydroxy(1-methylethyl)amino](oxo)acetate
-
-
metsulfuron-methyl
Mn2+
-
with acetolactate as substrate, Mn2+ behaves as a competitive inhibitor in presence of Mg2+
N'-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
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-
N'-(5-(2-fluorophenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-(2-methyl-phenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-(3-methyl-phenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-(3-pyridinyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
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N'-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
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N'-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-butyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
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N'-(5-ethyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
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-
N'-(5-furan-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-heptyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-iso-propyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-isopropyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-methyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
-
N'-(5-octyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-pentyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-phenyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N'-(5-propyl-1,3,4-thiadiazol-2-yl)-N-cyclopropyformyl-thiourea
-
-
N-(2-(piperidin-1-yl)ethyl)phthalimide
-
-
N-(3-bromophenyl)-1-cyanocyclopropanecarboxamide
-
inhibition rate: 17.25%
N-(3-chlorophenyl)-1-cyanocyclopropanecarboxamide
-
inhibition rate: 0%
N-(4-bromophenyl)-1-cyanocyclopropanecarboxamide
-
inhibition rate: 32.23%
N-(4-chlorophenyl)-1-cyanocyclopropanecarboxamide
-
inhibition rate: 93.92%
N-(5-substituted-1,3,4-thiadiazol-2-yl)-N-cyclopropylformyl-thiourea
-
-
N-Hydroxy-N-isopropyloxamate
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbic acid
-
5 mM, enhances activity 2-hydroxy-2-ethyl-3-oxobutanoate as substrate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
2-aceto-2-hydroxybutanoate
-
pH 8.2, 30C, chimeric enzyme Aabidopsis thaliana acetohydroxy acid synthase genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in E. coli
0.002 - 0.78
2-aceto-2-hydroxybutyrate
0.01 - 5.5
2-acetolactate
4.56
2-Ketobutyrate
-
pH 8.0, 37C, wild-type enzyme
6.91
2-ketoisovalerate
-
pH 8.0, 37C, wild-type enzyme
0.17
2-ketopantoate
-
pH 8.0, 37C, wild-type enzyme
3.15
2-Ketovalerate
-
pH 8.0, 37C, wild-type enzyme
0.21
3-hydroxy-2-ketobutyrate
-
pH 8.0, 37C, wild-type enzyme
0.27
3-hydroxy-3-methyl-2-ketobutyrate
-
pH 8.0, 37C, wild-type enzyme
0.334 - 15.3
3-hydroxypyruvate
0.893
Hydroxypyruvate
-
pH 8.0, 30C, recombinant enzyme
0.019 - 0.207
NADH
0.0089 - 0.072
NADP+
0.00016 - 11.2
NADPH
1.54
pyruvate
-
pH 8.0, 37C, wild-type enzyme
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00417
2,3-Dihydroxy-3-methylbutanoate
Escherichia coli
-
pH 7.4, 25C
78.3
2-aceto-2-hydroxybutyrate
Salmonella enterica subsp. enterica serovar Typhimurium
-
30C
1.8 - 18.3
2-acetolactate
0.167
2-Ketobutyrate
Escherichia coli
-
pH 8.0, 37C
0.182
2-ketoisovalerate
Escherichia coli
-
pH 8.0, 37C
0.194
2-ketopantoate
Escherichia coli
-
pH 8.0, 37C
0.05
2-Ketovalerate
Escherichia coli
-
pH 8.0, 37C
0.594
3-hydroxy-2-ketobutyrate
Escherichia coli
-
pH 8.0, 37C
3.511
3-hydroxy-3-methyl-2-ketobutyrate
Escherichia coli
-
pH 8.0, 37C
5.376
3-hydroxypyruvate
Escherichia coli
-
pH 8.0, 37C
0.00183 - 0.0883
NADH
0.00015 - 0.000667
NADP+
0.0000933 - 0.212
NADPH
0.021
pyruvate
Escherichia coli
-
pH 8.0, 37C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00002
1-aminocarbonyl-cyclopropane carboxylate
-
-
0.0203
1-aminocarbonylcyclopropanecarboxylate
-
pH 8.0, 30C, recombinant enzyme
0.031
1-carbamoylcyclopropanecarboxylic acid
-
pH 8.0, 30C
0.000058
1-cyano-cyclopropane carboxylate
-
-
207.9
1-cyano-N-[[(E)-(3,3-dichloroprop-1-yn-1-yl)diazenyl]sulfanyl]cyclopropanecarboxamide
-
-
0.0585
1-cyanocyclopropanecarboxylate
-
pH 8.0, 30C, recombinant enzyme
0.095 - 95.3
1-cyanocyclopropanecarboxylic acid
0.0056
1-hydroxycyclopropanecarboxylate
-
pH 8.0, 30C, recombinant enzyme
0.145
2,3-dihydroxy-3-isovalerate
-
pH 8.2, 30C, (2S)-2-aceto-2-hydroxybutanoate as variable substrate, 0.250 mM NADPH as fixed substrate
0.095 - 1.5
2,3-dihydroxyisovalerate
0.00019 - 0.00046
2-dimethylphosphinoyl-2-hydroxyacetic acid
0.24 - 3.6
2-hydroxy-2-methyl-3-oxopentanoate
0.35
4,4'-(pentamethylenedioxy)dibenzamidne bis(2-hydroxyethanesulfonate)
-
-
0.00009 - 0.0000903
cyclopropane-1,1-dicarboxylate
0.076
cyclopropane-1,1-dicarboxylic acid
-
pH 8.0, 30C
0.000716
dimethylmalonate
-
pH 8.0, 30C, recombinant enzyme
0.035
ethyl [(2-chlorophenyl)(hydroxy)amino](oxo)acetate
-
-
0.049
ethyl [hydroxy(2-methylphenyl)amino](oxo)acetate
-
-
0.351
ethyl [hydroxy(4-methylphenyl)amino](oxo)acetate
-
-
1.704
ethyl [[4-(cyanomethyl)phenyl](hydroxy)amino](oxo)acetate
-
-
0.0025 - 0.0032
ethylene glycol
0.034
methyl [hydroxy(1-methylethyl)amino](oxo)acetate
-
-
0.0027 - 0.00275
N-Hydroxy-N-isopropyloxamate
0.005 - 0.33
NADP+
0.0012 - 0.0226
NADPH
additional information
1-cyano-N-(4-methylphenyl)cyclopropanecarboxamide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.4
-
purified recombinant enzyme, substrate 2-acetolactate
1.97
-
purified recombinant enzyme, substrate hydroxypyruvate
3
-
deletion mutant DELTA423-430/F431S
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
-
constant activity with 2-acetolactate as substrate
7.5 - 8
-
reaction with 2-acetolactate, isoenzyme 1
8 - 8.5
-
reaction with 2-aceto-2-hydroxybutyrate
8.6
-
with 2-hydroxy-2-ethyl-3-oxobutanoate as substrate, Tris-HCl buffer, alpha-aceto-alpha-hydroxybutyrate as substrate, Tris buffer
9.4
-
reaction with 2,3-dihydroxyisovalerate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
-
pH 6.5: about 35% of maximal activity, pH 8.5: about 90% of maximal activity, reaction with 2-acetolactate, isoenzyme 1
6.6 - 8.8
-
pH 6.6: about 65% of maximal activity, pH 8.8: about 90% of maximal activity, reaction with 2-acetolactate, Tris buffer
6.8 - 8
-
pH 6.8: about 40% of maximal activity
7 - 8.5
7 - 9
-
pH 7.0: about 40% of maximal activity, pH 9.0: about 90% of maximal activity, with 2-hydroxy-2-ethyl-3-oxobutanoate as substrate, Tris-HCl buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 4.7
-
chromatofocusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
germinating, with appressoria, high expression level
Manually annotated by BRENDA team
additional information
-
ketol-acid reductoisomerase (Ilv5p), isocitrate lyase (Icl1p), fructose biphosphate aldolase (Fba1p) and pyruvate decarboxylase (Pdc2p) are present in the histatin-resistant derivative strain 132A but absent in the in the parent strain
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Alicyclobacillus acidocaldarius subsp. acidocaldarius (strain ATCC 27009 / DSM 446 / JCM 5260 / NBRC 15652 / NCIMB 11725 / NRRL B-14509 / 104-1A)
Azotobacter vinelandii (strain DJ / ATCC BAA-1303)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Ignisphaera aggregans (strain DSM 17230 / JCM 13409 / AQ1.S1)
Ignisphaera aggregans (strain DSM 17230 / JCM 13409 / AQ1.S1)
Ignisphaera aggregans (strain DSM 17230 / JCM 13409 / AQ1.S1)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Oryza sativa subsp. japonica
Oryza sativa subsp. japonica
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
-
monomeric mutant enzyme DELTA423-431/F431S, gel filtration
110000
-
dimeric wild-type enzyme, gel filtration
115000
205000
220000
228000
sedimentation equilibrium analysis
230000
gel filtration
235000
-
SDS-PAGE, isoenzyme 1
240000
-
Arabidopsis thaliana acetohydroxy acid synthase genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in E. coli, gel filtration
266000
-
native molecular mass
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 55000, SDS-PAGE
homohexamer
-
-
tetramer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
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purified enzyme contains 39-46% lipid
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with Mg2+ and NADPH at 2.3 A resolution. The binding of Mg2+ increases structural disorder while the binding of NADPH increases the structural rigidity of the enzyme. The binding of Mg2+ and NADPH opens the interface between the N- and C-domains, thereby allowing access for the substrates to bind
purified recombinant His-tagged enzyme, 9 mg/ml protein in 20 mM sodium HEPES, pH 7.5, and NADPH in a ratio of 10 mol NADPH per mol of enzyme, hanging drop vapour diffusion method, equal volumes of 0.003 ml of protein and reservoir solution, the latter containing 1.6 M ammonium sulfate, and 0.1 M sodium bicine, pH 9.0, 17C, 6 months, X-ray diffraction structure determination and analysis at 2.6 A resolution
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the enzyme is remarkably easy to crystallize
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enzyme KARI in complex with Mg2+ or with Mg2+ and NADPH, hanging-drop method by vaporphase diffusion at 18C, 0.003 ml of protein solution containing 6 mg/ml enzyme, 50 mM Hepes, pH 7.5, 5 mM NADPH, and 5 mM MgCl2, is mixed with 0.001 ml of reservoir solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M Tris-HCl, pH 8.5, and 15% w/v PEG 4000, a few days to 3 months, X-ray diffraction structure determination and analysis at 1.55 A and 2.80 A resolution, respectively, molecular replacement
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The dodecamer architecture of 23 point group symmetry is assembly of six dimeric units and dimerization is essential for the formation of the active site
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X-ray, structure analysis
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ammonium sulfate precipitation, crystal structure of the enzyme complexed with NADPH, two magnesium ions and N-hydroxy-N-isopropyloxamate, a herbicidal transition state analog determined at 1.65 A resolution, recombinant enzyme overexpressed in Escherichia coli
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crystallized with 2-aceto-2-hydroxybutanoate, Mn2+ and NADPH
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2
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40C, 5 min, 90% loss of activity
639171
6.5
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40C, 5 min, 55% loss of activity
639171
7
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40C, 5 min, 35% loss of activity
639171
7.5
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40C, 5 min, 30% loss of activity
639171
8
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40C, 5 min, 85% loss of activity
639171
8.5
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40C, 5 min, 90% loss of activity
639171
10
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rapid loss of activity above pH 10 with 2-acetolactate as substrate
639178
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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5 min, about 90% loss of activity at pH 6.2, about 55% loss of activity at pH 6.5, 35% loss of activity oH 7.0, 30% loss of activity at pH 7.5, about 85% loss of activity at pH 8.0, about 90% loss of activity at pH 8.5
additional information
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deletion mutant DELTA423-4301/F431S shows significantly lower temperature stability than wild type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
high activity loss during DEAE-cellulose chromatography, stable towards acetone fractionation and dialysis, unstable towards freezing and thawing
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unstable in absence of NADPH and Mg2+, very unstable in Tris buffer, 2-mercaptoethanol stabilizes
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10, in presence of NADPH, 5 d, 70-80% loss of activity
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-80, 20 mM MOPS, 10% glycerol, several months, mutant enzyme DELTA423-431/F431S, no loss of activity
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-80, chimeric enzyme composed of Arabidopsis thaliana acetohydroxy acid synthase genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in E. coli, several months, no loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant enzyme DELTA423-431/F431S; recombinant enzyme from Escherichia coli
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of wild-type and mutants
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partial
recombinant enzyme from Escherichia coli
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
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recombinant His6-tagged enzyme
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wild-type and mutant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressd in Escherichia coli and Saccharomyces cerevisiae
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expression as His6-tagged enzyme
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expression in Escherichia coli
expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene ilvC expression in Escherichia coli using an Escherichia coli-Corynebacterium glutamicum shuttle vector pECKA
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gene Pi-kari1, quantitative expression analysis
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mutant enzyme DELTA423-431/F431S, expression in Escherichia coli
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overexpression in Escherichia coli strain BL21(DE3)
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the nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase is genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D217E
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
D217N
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E213D
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75% reductoisomerase activity in comparison of wild-type enzyme
E213Q
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less than 4% reductoisomerase activity in comparison of wild-type enzyme
E221D
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E221Q
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E389D
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E389Q
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E393D
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
E393Q
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the mutant is insoluble, a soluble form is obtained only after denaturation
H132K
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less than 4% reductoisomerase activity in comparison of wild-type enzyme
H132Q
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less than 4% reductoisomerase activity in comparison of wild-type enzyme
K155E
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less than 4% reductoisomerase activity in comparison of wild-type enzyme
K155Q
-
less than 4% reductoisomerase activity in comparison of wild-type enzyme
K155R
-
less than 4% reductoisomerase activity in comparison of wild-type enzyme
K69L
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Km-value for NADP+ in the reaction with 2,3-dihydroxy-3-methylbutanoate is 2.1fold higher than the Km-value of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 163 compared to wild-type enzyme. Km-value for NADPH in the reaction with acetolactate is comparable to that of the wild-type emzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is 1.8fold higher than that of the wild-type enzyme
K75Q
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Km-value for NADP+ in the reaction with 2,3-dihydroxy-3-methylbutanoate is 2.7fold higher than the Km-value of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 77.5 compared to wild-type enzyme. Km-value for NADPH in the reaction with acetolactate is lower by a factor 2.9 compared to the Km-value of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 12.9 compared to wild-type enzyme
R68D/K69L/K75V/R76D
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turnover-number for reaction with NADH and acetolactate is 48fold higher compared to wild-type enzyme, turnover-number for reaction with NADPH and acetolactate is lower by factor 3.7 compared to wild-type enzyme, Km-value for NADH in the reaction with NADH and acetolactate is lower by a factor 10.8 compared to wild-type enzyme, Km-value for NADH in the reaction with NADPH and acetolactate is 30fold higher compared to wild-type enzyme
R68Q
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Km-value for NADP+ in the reaction with 2,3-dihydroxy-3-methylbutanoate is 6.9fold higher than the Km-value of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 345 compared to wild-type enzyme. Km-value for NADPH in the reaction with acetolactate is 3.4fold higher thahn that of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 18 compared to wild-type enzyme
R76D
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turnover-number for reaction with NADH and acetolactate is 48fold higher compared to wild-type enzyme, turnover-number for reaction with NADPH and acetolactate is lower by factor 4 compared to wild-type enzyme, Km-value for NADH in the reaction with NADH and acetolactate is lower by a factor 2.5 compared to wild-type enzyme, Km-value for NADPH in the reaction with NADPH and acetolactate is 55fold higher compared to wild-type enzyme
R76Q
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Km-value for NADP+ in the reaction with 2,3-dihydroxy-3-methylbutanoate is 17.1fold higher than the Km-value of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 258 compared to wild-type enzyme. Km-value for NADPH in the reaction with acetolactate is 5fold higher than that of the wild-type enzyme, the turnover-number for the reaction with NADP+ and 2,3-dihydroxy-3-methylbutanoate is lower by a factor 19.5 compared to wild-type enzyme
R76Q/R68A
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turnover-number for reaction with NADH and acetolactate is 20fold higher compared to wild-type enzyme, turnover-number for reaction with NADPH and acetolactate is lower by factor 28 compared to wild-type enzyme, Km-value for NADH in the reaction with NADH and acetolactate is comparable to that of wild-type enzyme, Km-value for NADPH in the reaction with NADPH and acetolactate is 22fold higher compared to wild-type enzyme
S414A
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
S414T
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less than 4% reductoisomerase activity in comparison to wild-type enzyme
DELTA1-17
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mutant with N-terminal 17-residue deletion: cellular localisation similar to wild-type, introduction into an ilv5DELTA strain, lacking the ilv5 gene, complements isoleucine and valine synthesis
DELTA1-33
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mutant with N-terminal 33-residue deletion: cellular localisation predominatly in the cytosol rather than in mitochondria. Deletion of the N-terminal 33 residues is sufficient to largely impair the protein's targeting to mitochondria. Introduction into an ilv5DELTA strain, lacking the ilv5 gene, complements isoleucine and valine synthesis
DELTA1-40
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mutant with N-terminal 40-residue deletion: cellular localisation throughout the cytosol rather than in mitochondria
DELTA1-46
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mutant with N-terminal 46-residue deletion: cellular localisation throughout the cytosol rather than in mitochondria. This mutant shows the largest steady-state protein level. Introduction into an ilv5DELTA strain, lacking the ilv5 gene, does not complements isoleucine and valine synthesis. Introduction into an industrial lager brewing strain, a robust expression of DELTA46 is as effective as that of a wild-type Ilv5p in lowering the total Vicinal diketones production in a 2l scale fermentation trial. Additional expression of DELTA46 does not alter the quality of the resultant beer in terms of contents of aromatic compounds and organic acids
DELTA1-53
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mutant with N-terminal 53-residue deletion: cellular localisation throughout the cytosol rather than in mitochondria
DELTA1-66
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mutant with N-terminal 66-residue deletion: weaker cytosolic localisation compared to other mutants. Mutant shows only a small amount of steady-state protein content
DELTA1-83
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mutant with N-terminal 83-residue deletion: weaker cytosolic localisation compared to other mutants. Mutant shows only a small amount of steady-state protein content
DELTA1-99
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mutant with N-terminal 99-residue deletion: weaker cytosolic localisation compared to other mutants. Mutant shows only a small amount of steady-state protein content
DELTA423-430/F431S
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mutant enzyme behaves as an active monomer with reduced thermal stability, KM-value for NADPH does not differ considerably from that for the wild-type enzyme, magnesium affinity is dramatically altered by monomerization
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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