1.1.1.85: 3-isopropylmalate dehydrogenase
This is an abbreviated version!
For detailed information about 3-isopropylmalate dehydrogenase, go to the full flat file.
Word Map on EC 1.1.1.85
Reaction
Synonyms
2-hydroxy-4-methyl-3-carboxyvalerate:NAD+ oxidoreductase, 2R,3S-isopropylmalate:NAD+ oxidoreductase (decaboxylating), 3-IPM dehydrogenase, 3-IPM-DH, 3-isopropylmalate dehydrogenase, 3-isopropylmalateDH, APS IPMDH, beta-IPM dehydrogenase, beta-IPMDH, beta-isopropylmalate dehydrogenase, beta-isopropylmalic enzyme, Bs3-isopropylmalateDH, dehydrogenase, 3-isopropylmalate, IMDH, IPMDH, IPMDH1, IPMDH2, IPMDH3, isopropylmalate dehydrogenase, leuB, NAD-dependent isopropylmalate dehydrogenase, Saci_0600, SbIPMDH, SoIPMDH, threo-Ds-3-isopropylmalate dehydrogenase, Tt-beta-3-isopropylmalateDH, two-domain 3-isopropylmalate dehydrogenase, Ydr417cp
ECTree
1.1.1.85 3-isopropylmalate dehydrogenaseAdvanced search results
results (
results (Engineering
Engineering on EC 1.1.1.85 - 3-isopropylmalate dehydrogenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D292N
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
F137L
mutation to corresponding residue of isoform IMPDH2, reduces activity toward 3-(2'-methylthio)-ethylmalate by 200fold, but enhances catalytic efficiency with 3-isopropylmalate to levels observed with isoform IPMDH2
L132A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
L133A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
L133F
mutation to corresponding residue of isoform IMPDH1, enhances catalytic efficiency with 3-(2'-methylthio)ethylmalate about100fold and reduces activity for 3-isopropylmalate
L134F
mutation to corresponding residue of isoform IMPDH1, reduces activity toward 3-(2'-methylthio)-ethylmalate, but enhances catalytic efficiency with 3-isopropylmalate
R136K
the mutant shows very strongly reduced catalytic efficiency compared to the wild type enzyme
R146A
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
R146K
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
R174K
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
V235A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
Y181A
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
Y181F
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
Y181H
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
E253A
-
specific activity: 4.7 unit/mg, Km: 0.016 mM (3-isopropylmalate), 0.246 mM (NAD), kcat: 14.9/sec
E253L
-
specific activity: 7.6 unit/mg, Km: 0.0122 mM (3-isopropylmalate), 0.474 mM (NAD), kcat: 13.7/sec
E253Q
-
specific activity: 7 unit/mg, Km: 0.0189 mM (3-isopropylmalate), 0.273 mM (NAD), kcat: 9.9/sec. this mutant is more thermostable than the other mutants
E253V
-
specific activity: 9 unit/mg, Km: 0.0244 mM (3-isopropylmalate), 0.171 mM (NAD), kcat: 12.4/sec
E253W
-
specific activity: 5.2 unit/mg, Km: 0.0254 mM (3-isopropylmalate), 0.244 mM (NAD), kcat: 8.3/sec
M256A
-
mutation increases thermostability by 6°C in comparison to wild-type
M256F
-
mutation decreases thermostability by 4°C in comparison to wild-type
M256V
-
mutation increases thermostability by 2°C in comparison to wild-type
D236R/D289K/I290Y/A296V/G337Y
-
engineering of the enzyme by site-directed mutagenesis in the cofactor binding pocket to utilize NADP+ as cofactor instead of NAD+, specificity change of factor 20000, modified cofactor binding structure, the mutant shows the same specificity for NADP+ as the wild-type enzyme for NAD+, overview
A266S
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
I268A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
M106L
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
A266S
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
-
I268A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
-
M106L
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
-
A268I
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
L106M
the mutant shows increased catalytic efficiency compared to the wild type enzyme
S266A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
A268I
Shewanella oneidensis MR-1 / ATCC 700550
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
-
L106M
Shewanella oneidensis MR-1 / ATCC 700550
-
the mutant shows increased catalytic efficiency compared to the wild type enzyme
-
S266A
Shewanella oneidensis MR-1 / ATCC 700550
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
-
A259S/F261P
Tm-value of mutant enzyme is 1.1°C higher than Tm-value of wild-type enzyme
F145S
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
F3S
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
F3S/L288P
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme. The MA153 strain containing a doubly mutated leuB gene grows more rapidly than does Escherichia coli MA153 containing the wild-type leuB gene in leucine-free M9 minimal liquid medium
F3S/S232G
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme. The MA153 strain containing a doubly mutated leuB gene grows more rapidly than does Escherichia coli MA153 containing the wild-type leuB gene in leucine-free M9 minimal liquid medium
G154A
Tm-value of mutant enzyme is 1.9°C higher than Tm-value of wild-type enzyme
I68L/I142T/K335E
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
K152R
Tm-value of mutant enzyme is 0.4°C lower than Tm-value of wild-type enzyme
K152R/G154A
Tm-value of mutant enzyme is 1.2°C higher than Tm-value of wild-type enzyme
L288P
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
L328S
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
L336P
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
M91L/I95L
Tm-value of mutant enzyme is 3.2°C higher than Tm-value of wild-type enzyme
M91L/I95L/K152R/G154A/A259S/F261P/Y282L
Tm-value of mutant enzyme is 0.4°C higher than Tm-value of wild-type enzyme
R284G
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
Y282L
Tm-value of mutant enzyme is 0.3°C lower than Tm-value of wild-type enzyme
Y309H
random mutagenesis, the mutation alters the Km values for NAD+ and/or the kcat value, the mutant shows reduced thermostability compared to the wild-type enzyme
G154A
-
Tm-value of mutant enzyme is 1.9°C higher than Tm-value of wild-type enzyme
-
K152R
-
Tm-value of mutant enzyme is 0.4°C lower than Tm-value of wild-type enzyme
-
K152R/G154A
-
Tm-value of mutant enzyme is 1.2°C higher than Tm-value of wild-type enzyme
-
M91L/I95L
-
Tm-value of mutant enzyme is 3.2°C higher than Tm-value of wild-type enzyme
-
M91L/I95L/K152R/G154A/A259S/F261P/Y282L
-
Tm-value of mutant enzyme is 0.4°C higher than Tm-value of wild-type enzyme
-
A172D
A172F
A172G
-
melting temperature is reduced by 0.3°C compared to wild-type enzyme, crystal structure is similar to that of the wild-type enzyme
A172I
-
melting temperature is increased by 2°C compared to wild-type enzyme
A172L
A172V
A31G/G43A/A709G
-
optimum temperature is shifted to lower temperatures compared to wild-type enzyme, activity is less temperature dependent than that of the wild-type, resulting in higher activity at temperatures below 60°C, improved turnover-numbers at 40°Cm increase in Km-values, slight loss of thermal stability
A335E
C275T
-
similar pH-dependence to that of the wild-type enzyme, improved binding affinities for both D-3-isopropylmalate and NAD+ result in an improved catalytic efficiency, mutant enzyme retains thermal stability
D184H
D217A
the mutant retains 1.1% of the catalytic activity of the wild type enzyme
D241A
the mutant shows a drastic decrease in the kcat value (0.06% compared to that of the wild type enzyme)
D245A
the mutant retains 10.5% of the catalytic activity of the wild type enzyme
E270A
E57V/R85M/Y86A/M208T/F217Y/V238M/R310M
no (2R,3S)-3-isopropylmalate dehydrogenase activity detected
E57V/S72I/R85M/Y86A/F217Y/V238M/R310M
no (2R,3S)-3-isopropylmalate dehydrogenase activity detected
E57V/S72I/R85M/Y86A/M208T/F217Y/R310M
in comparison to mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M this mutant shows a significant decrease in kcat ((2R,3S)-3-isopropylmalate), alteration of Km value only moderate
E57V/S72I/R85M/Y86A/M208T/F217Y/V238M
in comparison to mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M this mutant shows a significant decrease in kcat ((2R,3S)-3-isopropylmalate), alteration of Km value only moderate
E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M
in comparison to mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M this mutant shows a larger kcat ((2R,3S)-3-isopropylmalate) and a larger Km((2R,3S)-3-isopropylmalate) or (NAD+) temperatures giving half of the initial activity is 87.8 °C
E57V/S72I/R85M/Y86A/M208T/V238M/R310M
in comparison to mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M this mutant shows a significant decrease in kcat ((2R,3S)-3-isopropylmalate), alteration of Km value only moderate
E57V/S72I/R85V/Y86A/M208T/F217Y/V238M/R310M
no (2R,3S)-3-isopropylmalate dehydrogenase activity detected
E87G
-
mutation does not affect the dissociation constant from the binary complex, but does greatly increase the KM-values
E87Q
-
mutation does not affect the dissociation constant from the binary complex, but does greatly increase the KM-values
F53L
G376A
-
optimum temperature is shifted to lower temperatures compared to wild-type enzyme, activity is less temperature dependent than that of the wild-type, resulting in higher activity at temperatures below 60°C, improved turnover-numbers at 40°C, increase in Km-values, mutant enzyme retains thermal stability
G43A
-
optimum temperature is shifted to lower temperatures compared to wild-type enzyme, activity is less temperature dependent than that of the wild-type, resulting in higher activity at temperatures below 60°C, improved turnover-numbers at 40°C, increase ion Km-values, slight loss of thermal stability
H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M
TtHICDH variant with considerably higher IPMDH activity than double mutant R85V/Y86A, Km ((2R,3S)-3-isopropylmalate) or (NAD+) lower than wild-type HICDH but still considerably higher than wild-type (2R,3S)-3-isopropylmalate dehydrogenase, kcat ((2R,3S)-3-isopropylmalate) much higher than wild-type HICDH but still considerably lower than wild-type (2R,3S)-3-isopropylmalate dehydrogenase, temperatures giving half of the initial activity is 79.9 °C, crystal structure determined at 2.4 A, mutant is a homotetramer
H179K
K185A
the mutant shows a drastic decrease in the kcat value (0.06% compared to that of the wild type enzyme)
L134N
L134N/V181T/P324T/A335E
site-directed mutagenesis, exchange of residues for those of ancestral mutants, the mutant shows increased thermal stability compared to the wild-type enzyme, the mutant shows increased thermal stability compared to the wild-type enzyme
L204F
-
higher thermostability compared to wild-type enzyme, denaturation rates at 68°C and 70°C are slower
N102A
the mutant retains 13% of the catalytic activity of the wild type enzyme
P324T
P56E
R58L
R85V/Y86A
TtHICDH variant with high IPMDH activity, Km ((2R,3S)-3-isopropylmalate) or (NAD+) lower than wild-type HICDH but still considerably higher than wild-type (2R,3S)-3-isopropylmalate dehydrogenase, kcat ((2R,3S)-3-isopropylmalate) higher than wild-type HICDH but still considerably lower than wild-type (2R,3S)-3-isopropylmalate dehydrogenase, temperatures giving half of the initial activity is 92.5 °C
S226R
-
Km-value for NADP+ is about 40% compared to wild-type enzyme, Km-value for NAD+ is 2.6fold higher compared to wild-type enzyme
S226R/D278K/I279Y
-
Km-value for NADP+ is about 0.8% compared to wild-type enzyme, Km-value for NAD+ is 153fold higher compared to wild-type enzyme
S261N
S72I/R85M/Y86A/M208T/F217Y/V238M/R310M
in comparison to mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M this mutant shows a significant decrease in kcat ((2R,3S)-3-isopropylmalate), alteration of Km value only moderate
T16V
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
V126A
-
the mutation decreases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126E
-
the mutation increases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126F
-
the mutation increases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126G
-
the mutation decreases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126I
-
the mutation increases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126L
-
the mutation increases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126M
-
the mutation increases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126S
-
the mutation decreases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V126T
-
the mutation decreases the KM value of DL-3-isopropylmalate at 60°C in comparison to wild-type
V181T
V181T/P324T/A335E
site-directed mutagenesis, exchange of residues for those of ancestral mutants, the mutant highly shows increased thermal stability compared to the wild-type enzyme, the mutant shows decreased thermal stability compared to the wild-type enzyme
V61I
W152F
site-directed mutagenesis, the W152F mutation causes a significant decrease in the amplitude of only the slow part of refolding, without influencing the amplitude of the burst part, the mutant shows an altered tertiary structure compared to the wild-type enzyme conformation
W152F/W195F
site-directed mutagenesis, the W152F mutation causes a significant decrease in the amplitude of only the slow part of refolding, without influencing the amplitude of the burst part, the mutant shows an altered tertiary structure compared to the wild-type enzyme conformation
W77F/W152F
site-directed mutagenesis, the W152F mutation causes a significant decrease in the amplitude of only the slow part of refolding, without influencing the amplitude of the burst part, the mutant shows an altered tertiary structure compared to the wild-type enzyme conformation
Y139A
the mutant retains 2.9% of the catalytic activity of the wild type enzyme
A172L
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
the mutant shows improved thermal stability compared to the wild type enzyme
-
E270A
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
the mutant exhibits largely reduced (about 1%) catalytic activity and negligible activation by K+ compared to the wild type enzyme
-
additional information
A172D
-
melting temperature is reduced by 2.7°C compared to wild-type enzyme
A172F
-
mutation causes rearrangement in domain structure, which leads to a higher thermostability compared to wild-type enzyme
A172F
-
melting temperature is increased by 1.8°C compared to wild-type enzyme
A172L
-
mutation causes rearrangement in domain structure, which leads to a higher thermostability compared to wild-type enzyme
A172L
-
melting temperature is increased by 3°C compared to wild-type enzyme
A172L
the mutant shows improved thermal stability compared to the wild type enzyme
A172V
-
melting temperature is increased by 1°C compared to wild-type enzyme, crystal structure is similar to that of the wild-type enzyme
A172V
mutation improves thermal stability, without significantly changing its catalytic properties
A335E
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
A335E
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased thermal stability compared to the wild-type enzyme, the mutant shows increased thermal stability compared to the wild-type enzyme
D184H
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
D184H
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased catalytic efficiency with NAD+ compared to the wild-type enzyme
E270A
the mutant exhibits largely reduced (about 1%) catalytic activity and negligible activation by K+ compared to the wild type enzyme
E270A
the mutation leads to a significant increase in the Km value for NAD+ compared to the wild type enzyme
F53L
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows decreased thermal stability compared to the wild-type enzyme
F53L
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased catalytic efficiency with NAD+ compared to the wild-type enzyme
H179K
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows decreased thermal stability compared to the wild-type enzyme
H179K
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased catalytic efficiency with NAD+ compared to the wild-type enzyme
L134N
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
L134N
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased thermal stability compared to the wild-type enzyme, the mutant shows highly increased thermal stability compared to the wild-type enzyme
P324T
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
P324T
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased thermal stability compared to the wild-type enzyme, the mutant shows increased thermal stability compared to the wild-type enzyme
P56E
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
P56E
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased catalytic efficiency with NAD+ compared to the wild-type enzyme
R58L
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows decreased thermal stability compared to the wild-type enzyme
R58L
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows increased catalytic efficiency with NAD+ compared to the wild-type enzyme
S261N
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows decreased thermal stability compared to the wild-type enzyme
S261N
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows slightly increased catalytic efficiency with NAD+ compared to the wild-type enzyme
V181T
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows increased thermal stability compared to the wild-type enzyme
V181T
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows decreased thermal stability compared to the wild-type enzyme, the mutant shows decreased thermal stability compared to the wild-type enzyme
V61I
site-directed mutagenesis, exchange of a cofactor binding residue to the residue of the ancestral mutant, the mutant shows decreased thermal stability compared to the wild-type enzyme
V61I
site-directed mutagenesis, exchange of a residue for that of ancestral mutants, the mutant shows slightly increased catalytic efficiency with NAD+ compared to the wild-type enzyme
additional information
-
results show that, for residues with non-polar side chains, when positioned at residue 253, the thermostabilities of their corresponding mutants correlate positively with the relative hydrophobicities of the side chains
additional information
-
two ancestral sequences of 3-isopropylmalate dehydrogenase are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences are artificially synthesized and expressed in Escherichia coli. The ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions
additional information
designing thermostable 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree and ancestral mutant sequences, overview
additional information
-
designing thermostable 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree and ancestral mutant sequences, overview
additional information
one or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase is/are substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contains three amino acid substitutions, shows a 17fold higher specific activity at 25°C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant show similar patterns along the reaction coordinate to those of the mesophilic enzyme
additional information
-
one or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase is/are substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contains three amino acid substitutions, shows a 17fold higher specific activity at 25°C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant show similar patterns along the reaction coordinate to those of the mesophilic enzyme
