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.

Reaction

(2R,3S)-3-isopropylmalate
+
NAD+
=
(2S)-2-isopropyl-3-oxosuccinate
+
NADH
+
H+

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 Oxidoreductases
         1.1 Acting on the CH-OH group of donors
             1.1.1 With NAD+ or NADP+ as acceptor
                1.1.1.85 3-isopropylmalate dehydrogenase

Engineering

Engineering on EC 1.1.1.85 - 3-isopropylmalate dehydrogenase

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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
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
E253F
-
specific activity: 7.6 unit/mg
E253G
-
specific activity: 5.9 unit/mg
E253I
-
specific activity: 7.4 unit/mg
E253K
-
when a lysine is the position 253 the protein dissociates
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
M256I
-
no effect
M256L
-
no effect
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
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
-
A172E
-
enhanced thermostability compared to wild-type enzyme
A172F
-
melting temperature is increased by 1.8°C compared to wild-type enzyme; mutation causes rearrangement in domain structure, which leads to a higher thermostability compared to wild-type enzyme
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
A172N
-
enhanced thermostability compared to wild-type enzyme
A172Q
-
enhanced thermostability compared to 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
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
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
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
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
G240A
-
decreased thermostability
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
K185A
-
the mutant shows a drastic decrease in the kcat value (0.06% compared to that of the wild type enzyme)
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
L246E/V249M
-
decreased thermostability
N102A
-
the mutant retains 13% of the catalytic activity of the wild type enzyme
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
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/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
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
additional information