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1.8.1.4: dihydrolipoyl dehydrogenase

This is an abbreviated version!
For detailed information about dihydrolipoyl dehydrogenase, go to the full flat file.

Word Map on EC 1.8.1.4

Reaction

protein N6-(dihydrolipoyl)lysine
+
NAD+
=
protein N6-(lipoyl)lysine
 +
NADH
 +
H+

Synonyms

apicoplast E3, CDS, CIP50, coronin-interacting protein, dehydrogenase, lipoamide, dehydrolipoate dehydrogenase, DHLDH, DHLipDH, diaphorase, dihydrolipoamide dehydrogenase, dihydrolipoamide dehydrogenase E3, dihydrolipoamide:NAD+ oxidoreductase, dihydrolipoic dehydrogenase, dihydrolipomide dehydrogenase, dihydrolipoyl dehydrogenase, DLD, DLD1, Dld2, DLDH, DLDH dehydrogenase, DLDH diaphorase, DLDH2, DT-diaphorase, E3, E3 component, E3 component of 2-oxoglutarate dehydrogenase complex, E3 component of acetoin cleaving system, E3 component of alpha keto acid dehydrogenase complexes, E3 component of pyruvate and 2-oxoglutarate dehydrogenases complexes, E3 component of pyruvate complex, E3 lipoamide dehydrogenase, E3 protein component of 2-oxoacid dehydrogenase multienzyme complexes, E3 subunit of the alpha-ketoglutarate dehydrogenase complex, EC 1.6.4.3, Glycine cleavage system L protein, Glycine oxidation system L-factor, hDLDH, hE3, hLADH, L-protein, LAD, LADH, LDH, LDP-Glc, LDP-Val, LipDH, lipoamide dehydrogenase, lipoamide dehydrogenase (NADH), lipoamide dehydrogenase C, lipoamide dehydrogenase2, lipoamide oxidoreductase (NADH), lipoamide reductase, lipoamide-dehydrogenase-valine, lipoate dehydrogenase, lipoic acid dehydrogenase, lipoyl dehydrogenase, LPD, LPD-GLC, LPD-VAL, LPD1, LPD2, Lpd3, LpdA, LpdC, LpdG, LpdV, More, mtLPD2, NAD(P)H:lipoamide oxidoreductase, NADH dehydrogenase, NADH diaphorase, NADH:lipoamide oxidoreductase, nicotinamide adenine dinucleotide diaphorase, ORF-E3, pdhD, pdhL, PfaE3, rhDLDH, TAase, ubiquinone reductase

ECTree

     1 Oxidoreductases
         1.8 Acting on a sulfur group of donors
             1.8.1 With NAD+ or NADP+ as acceptor
                1.8.1.4 dihydrolipoyl dehydrogenase

Engineering

Engineering on EC 1.8.1.4 - dihydrolipoyl dehydrogenase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C44S
-
0.003% of the activity of wild-type enzyme with NAD+ and dihydrolipoamide. Enzyme is capable to catalyze reactions with NADH as electron donor and ferricyanide, thio-NAD+, 2,6-dichlorophenol indophenol and O2 as electron acceptor. The fluorescence of FAD in oxidized wild-type enzyme is markedly temperature dependent, while the fluorescence of FAD in mutants C44S and C49S is independent of temperature
C49S
-
0.012% of the activity of wild-type enzyme with NAD+ and dihydrolipoamide. Enzyme is capable to catalyze reactions with NADH as electron donor and ferricyanide, thio-NAD+, 2,6-dichlorophenol indophenol and O2 as electron acceptor. The fluorescence of FAD in oxidized wild-type enzyme is markedly temperature dependent, while the fluorescence of FAD in mutants C44S and C49S is independent of temperature
K53R
-
spectral and redox properties of FAD in the mutant enzyme as well as the interaction of the flavin with bound NAD+ are profoundly affected by the mutation, K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of reducing substrate. The absorbance spectrum in the visible and near-ultraviolet is little changed from that of wild-type enzyme, in contrast to wild-type enzyme the spectrum of K53R is sensitive to pH. Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum
E354K
-
is significantly less sensitive to NADH inhibition than the native LPD
H322Y
-
upon purification LPD loses activity and associated FAD at the gel filtration step
E423A
in 2 M KCl, the mutant is significantly less active than wild-type, decreased Km value for dihydrolipoamide. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
E423D
wild-type and E423D mutant enzyme are much less active in the absence of KCl than in its presence. The mutant enzyme is inactivated at temperatures around 20°C lower than the wild-type
E423Q
in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
E423S
in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
E423A
-
in 2 M KCl, the mutant is significantly less active than wild-type, decreased Km value for dihydrolipoamide. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
-
E423D
-
wild-type and E423D mutant enzyme are much less active in the absence of KCl than in its presence. The mutant enzyme is inactivated at temperatures around 20°C lower than the wild-type
-
E423Q
-
in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
-
E423S
-
in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme
-
A1444G
-
substitution located in exon 13 leading to 20% of wild type activity
A328V
site-directed mutagenesis of the conserved residue, the site-specific dihydrolipoamide dehydrogenase mutant shows a switched kinetic mechanism, it shows a random sequential kinetic mechanism with an interaction factor (alpha) of 8.5. The mutation deteriorates substantially the catalytic power of human E3 enzyme increasing the binding affinity for NAD+ and dihydrolipoamide . The mutation triggers this potential intrinsic property of the enzyme causing the kinetic mechanism of the mutant to switch from a ping-pong mechanism to a random sequential mechanism
C45S
-
Ser-45 mutant is highly purified, shows 5270fold lower activity than wild-type enzyme. Destroyed disulfide bond between Cys-45 and Cys-50 of the active disulfide center in human E3. UV-visible spectrum of the Ser-45 mutant is similar to that of the reduced form of the enzyme and the second fluorescence emission of the mutant disappears
C45Y
-
purification of the Tyr-45 mutant is not successful. Recombinant human E3 becomes too unstable to be easily obtained from Escherichia coli
C50A
site-directed mutagenesis, catalytic efficiency of mutant C50A toward NAD+ decreases 5317fold compared to the wild-type enzyme, the mutation destroys the active disulfide center between Cys45 and Cys50, which restricts the freedom of Cys50
C50T
site-directed mutagenesis, catalytic efficiency of mutant C50A toward NAD+ decreases 2057fold compared to the wild-type enzyme, the mutation destroys the active disulfide center between Cys45 and Cys50, which restricts the freedom of Cys50
D320N
D413A
D413N
-
the mutant shows 119% activity in the forward reaction and 96% activity in the reverse reaction compared to the wild type enzyme
D444V
D473L
-
site-directed mutagenesis, mutant shows about 37fold decreased activity and small conformational changes compared to the wild-type enzyme
DELTAG101
naturally occuring mutation, the mutation is involved in E3 deficiency
E192Q
-
specific activity is markedly decreased, less than 5% of the wild-type activity, Km-values for lipoamide and dihydrolipoamide are markedly reduced
E340K
E431A
-
exhibits very similar expression levels and purification yields as the wild-type, but abolishes the proteolytic activity
E457Q
-
molar ratio of FAD to enzyme is 0.9 compared to 1 for the wild-type enzyme, mutation affects the environment surrounding FAD, decrease in efficiency of electron transfer from the reduced flavin to the oxidized substrate
G194C
G426E
naturally occuring mutation, the mutation is involved in E3 deficiency
H329A
site-directed mutagenesis, the kcat value of the mutant is significantly decreased by 24fold as compared to the wild-type, indicating that the mutation severely deteriorates the catalytic power of the enzyme
H348A
-
the mutant shows 60% activity in the forward reaction and 66% activity in the reverse reaction compared to the wild type enzyme
H348L
-
the mutant shows 65% activity in the forward reaction and 74% activity in the reverse reaction compared to the wild type enzyme
H450A
-
shows an increase in proteolytic activity as compared with the wild-type
H452Q
-
molar ratio of FAD to enzyme is 0.94 compared to 1 for the wild-type enzyme, no production of NADH when the enzyme is reduced by dihydrolipoamide, transfer of electrons from the substrate dihydrolipoamide to NAD+ is extremely low
I12T
naturally occuring mutation, the mutation is involved in E3 deficiency
I318T
naturally occuring mutation, the substitution triggers major structural disturbance only at the C-terminus
I358T
-
mutation affecting the ability of FAD, NAD+, or NADH to bind to E3
I445M
I51A
-
mutant with about 100fold reduced activity compared to the wild type enzyme
K54E
-
about 25% less bound FAD compared to wild-type, specific activity is markedly decreased, less than 5% of the wild-type activity, Km-value for lipoamide is increased by about twofold
L99A
the mutation deteriorates the catalytic power of the enzyme substantially
M326V
N286D
N286Q
P154A
the mutation makes enzyme binding to both dihydrolipoamide and NAD+ inefficient
P156A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme
P303A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme
P325A
-
mutation of highly conserved resdue in the central domain, about 150fold decrease in kcat value
P355A
the mutation makes enzyme binding to NAD+ substantially less efficient. The catalytic efficiency of the mutant toward NAD+ is decreased by 81% compared to the wild type enzyme
P387A
the mutation deteriorates severely the catalytic power of the enzyme
P423A
the mutation makes enzyme binding to both dihydrolipoamide and NAD+ inefficient
P453L
P453V
-
1650fold lower specific activity compared to the wild type enzyme
R281K
specific activity is 11.93% to that of wild-type E3. FAD-content is about 93% that of wild-type E3. Kcat of forward reaction is decreased dramatically. Substitution has no effect in the self-dimerization
R281N
specific activity is 12.50% to that of wild-type E3. FAD-content is about 96% that of wild-type E3. Kcat of forward reaction is decreased dramatically. Substitution has no effect in the self-dimerization
R447A
-
the mutant shows 110% activity in the forward reaction and 122% activity in the reverse reaction compared to the wild type enzyme
R447G
R460G
S456A
-
exhibits very similar expression levels and purification yields as the wild-type, but abolishes the proteolytic activity
S456A/D444V
-
low levels of residual activity
S53K/K54S
-
about 25% less bound FAD compared to wild-type, specific activity is markedly decreased, less than 5% of the wild-type activity, Km-values for lipoamide and dihydrolipoamide are markedly reduced. The catalytic rate constant, turnover number/Km, is significantly lower than wild-type
T148G
specific activity is 76.34% to that of wild-type E3. FAD-content is about 710% that of wild-type E3. Substitution has no effect in the self-dimerization
T148S
specific activity is 88.62% to that of wild-type E3. FAD-content is about 92% that of wild-type E3. Substitution has no effect in the self-dimerization
T44V
-
site-directed mutagenesis, mutation of Thr44 of the FAD-binding region to Val, corresponding to the prokaryotic sequence, results in 2.2fold reduced activity with a slightly different microenvironment at the FAD-binding site
W366A
-
mutation of highly conserved residue. kinetic parameters similar to wild-type
Y438A
Y438F
-
the mutant shows 100% activity in the forward reaction and 112% activity in the reverse reaction compared to the wild type enzyme
Y438H
A48I
-
the mutation decreases the Km for dihydrolipoamide substrate by 3fold compared to the wild type enzyme
A54I
-
the mutation increases the Km for dihydrolipoamide substrate by 1fold and NAD+ by 3fold compared to the wild type enzyme
C15T
-
the mutation increases the Km for dihydrolipoamide substrate by 5fold and NAD+ by 3fold compared to the wild type enzyme
C38G
-
the mutation increases the Km for NAD+ by 9fold without affecting Km for dihydrolipoamide compared to the wild type enzyme
Cys38Gly
-
the substitution does not show any change in Km value for dihydrolipoamide compared to the wild type enzyme
D49G
-
the mutation decreases the Km for dihydrolipoamide substrate by 2fold compared to the wild type enzyme
A181V
the mutant is not inhibited by N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid compared to the wild type enzyme
A290R
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
D5A
4.3% activity of the wild type enzyme
E91A
49.6% activity of the wild type enzyme
E91K
62.5% activity of the wild type enzyme
F269R
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
F464A
5% activity of the wild type enzyme
G312A/L313G/L314P/Q315M
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
H386A
20% activity of the wild type enzyme
H386K
9% activity of the wild type enzyme
H460E
69.4% activity of the wild type enzyme
H98A
3.3% activity of the wild type enzyme
K103E
17.7% activity of the wild type enzyme
K105A
44.7% activity of the wild type enzyme
K216A
58.3% activity of the wild type enzyme
K220A
81.2% activity of the wild type enzyme
K223A
67.1% activity of the wild type enzyme
K223E
70.5% activity of the wild type enzyme
K224A
55.2% activity of the wild type enzyme
K376A
52.2% activity of the wild type enzyme
K67A
67.3% activity of the wild type enzyme
K67E
64.3% activity of the wild type enzyme
K88E
74.8% activity of the wild type enzyme
L314P
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
N209V
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
N43A
11% activity of the wild type enzyme
R147T
the mutant is more sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
R347S
the mutant is not inhibited by N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid compared to the wild type enzyme
R93A
6.5% activity of the wild type enzyme
R93E
3.6% activity of the wild type enzyme
A181V
-
the mutant is not inhibited by N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid compared to the wild type enzyme
-
A290R
-
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
-
F269R
-
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
-
N209V
-
the mutant is less sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
-
R147T
-
the mutant is more sensitive to N-(2,4-dichlorophenethyl)-2-[8-(2,4-dimethoxybenzoyl)-4-oxo-1-phenyl-1,3,8-triazaspiro-[4.5]decan-3-yl]acetamid than the wild type enzyme
-
E91A
-
49.6% activity of the wild type enzyme
-
K220A
-
81.2% activity of the wild type enzyme
-
K223A
-
67.1% activity of the wild type enzyme
-
K224A
-
55.2% activity of the wild type enzyme
-
K67A
-
67.3% activity of the wild type enzyme
-
I192G
site-directed mutagenesis, the mutant is active with phenazine-1-carboxylic acid
V191Y
site-directed mutagenesis, the mutant is active with phenazine-1-carboxylic acid
I192G
-
site-directed mutagenesis, the mutant is active with phenazine-1-carboxylic acid
-
V191Y
-
site-directed mutagenesis, the mutant is active with phenazine-1-carboxylic acid
-
K43A
-
unable to express the mutant protein
K43R
-
expresses well, but still has lipoic acid attached
additional information