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C243S
site-directed mutagenesis, the mutant shows increased specific activity, the mutation at Cys243 does not significantly affect ADH kinetic efficiency
C47S
site-directed mutagenesis, the mutation Ser causes an almost complete loss of the enzyme activity
H51Q
site-directed mutagenesis, shifting of pH dependency, increased activity at pH 8.0, decrease of the rate of isomerization of the enzyme-NAD+ complex, which becomes the limiting step for ethanol oxidation
H51Q/K228R
site-directed mutagenesis, kinetic effects
W54L
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less active than the wild-type enzyme with ethanol, 1-propanol and 1-butanol. With 1-pentanol and 1-hexanol the mutant enzyme is a better catalyst than the wild-type enzyme
C257L
mutation introduced to improve stability under oxidzing conditions. Mutant exhibits prolonged stability and an elevated inactivation temperature
V260A
kinetic parameters and temperature dependencies similar to wild-type
W49F/W167Y
kinetic parameters and temperature dependencies similar to wild-type
W49F/W167Y/V260A
kinetic parameters and temperature dependencies similar to wild-type
W49F/W87F
kinetic parameters and temperature dependencies similar to wild-type
W49F/W87F/V260A
kinetic parameters and temperature dependencies similar to wild-type
W87A
mutation results in a loss of the Arrhenius break seen at 30°C for the wild-type enzyme and an increase in cold lability due to destabilization of the active tetrameric form. Kinetic isotope effects are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30°C for the wild-type enzyme
W87F
investigation on protein dynamics on the microsecond time scale. Mutant exhibits a fast, temperature-independent microsecond decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results rule out an ionizing histidine as the origin of the fluorescence quenching. A Trp49-containing dimer interface may act as a conduit for thermally activated structural change within the protein interior
W87F/H43A
investigation on protein dynamics on the microsecond time scale. Mutant exhibits a fast, temperature-independent microsecond decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results rule out an ionizing histidine as the origin of the fluorescence quenching. A Trp49-containing dimer interface may act as a conduit for thermally activated structural change within the protein interior
Y25A/W49F/W167Y
kinetic parameters and temperature dependencies similar to wild-type
Y25A/W49F/W167Y/V260A
kinetic parameters and temperature dependencies similar to wild-type
Y25A/W49F/W87F
kinetic parameters and temperature dependencies similar to wild-type
Y25A/W49F/W87F/V260A
kinetic parameters and temperature dependencies similar to wild-type
A93F
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isozyme alphaalpha, altered active site structure and inhibitor binding
S48T
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isozyme gamma(2)gamma(2), altered active site structure and inhibitor binding
V141L
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isozyme gamma(2)gamma(2), altered active site structure and inhibitor binding
P47A
site-directed mutagenesis, about 100fold increased activity compared to the wild-type enzyme
P47H
site-directed mutagenesis, about 100fold increased activity compared to the wild-type enzyme
P47Q
site-directed mutagenesis, about 100fold increased activity compared to the wild-type enzyme
G223D
-
unaltered cofactor specificity compared to the wild-type enzyme
G223D/T224I
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highly reduced activity with NADP+ compared to the wild-type enzyme, wild-type-like activity with NAD+
G223D/T224I/H225N
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altered cofactor specificity, highly reduced activity with NADP+ compared to the wild-type enzyme, wild-type-like activity with NAD+
H225N
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unaltered cofactor specificity compared to the wild-type enzyme
T224I
-
unaltered cofactor specificity compared to the wild-type enzyme
G211C
-
mutant enzyme exhibits almost complete reversals in cofactor specificity
G211InsA
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with NADP+, the mutant enzyme shows a 29-fold increase in kcat as compared to the wild-type enzyme
G211InsC
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with NAD+, the mutant enzyme exhibits 6-fold decrease in kcat as compared to wild-type enzyme. Mutant enzyme exhibits almost complete reversals in cofactor specificity
G211InsG
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with NAD+, the mutant enzyme exhibits 2.5-fold enhancement in kcat over the wild-type enzyme. Activity with NADP+ exceeds that of the wild type enzyme
G211InsS
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with NAD+, the mutant enzyme exhibits 2.5-fold enhancement in kcat over the wild-type enzyme. With NADP+, the mutant enzyme shows a 29-fold increase in kcat as compared to the wild-type enzyme
G211S
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with NAD+, the mutant enzyme exhibits 1.5-fold enhancement in kcat over the wild-type. Activity with NADP+ exceeds that of the wild type enzyme
H255R
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the H255R single mutant exhibits an increased binding affinity toward NADP+ and a concomitant reduction in affinity for NAD+. The apparent kcat for H255R is about 60% of that of the wild-type with NAD+, but it is sixfold higher than the wild-type with NADP+. Position 255 is important for recognizing NADP(H), but is not the sole determinant of cofactor specificity
K249G
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catalytic efficiency increases 5fold for NADH, the efficiency with NADPH increases more than 30fold in comparison to the wild-type
K249G/H255R
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the catalytic efficiency with NADH increases 4fold, the efficiency with NADPH increases more than 16fold. In the oxidation reaction, the kcat with NAD+ improves by 15fold for the double mutant over the wild type enzyme. With NADP+ the kcat is nearly two orders of magnitude larger than the wild-type. Mutant exhibits significantly improved activity and broadened cofactor specificity as compared to the wild-type enzyme
F43H
-
site-directed mutagenesis, mutant C1 variant
F43H/Y54L
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site-directed mutagenesis, mutant C1B1 variant
F43T/Y54G/L119Y/F282W
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site-directed mutagenesis, mutant B1F4 variant
H39Y/F43H/Y54F/Y294F/W295A
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site-directed mutagenesis, mutant A2C2B1 variant
H39Y/F43S/Y294F/W295A
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site-directed mutagenesis, mutant A2C3 variant
W295A
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site-directed mutagenesis, mutant A1 variant
Y54G/L119Y
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site-directed mutagenesis, mutant B1 variant
H39Y/F43H/Y54F/Y294F/W295A
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site-directed mutagenesis, mutant A2C2B1 variant
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W295A
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site-directed mutagenesis, mutant A1 variant
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E97C
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shows the same activity but a reduced thermostability with respect to the wild type recombinant protein
W95L
the mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme, the substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure, optimum pH is at about pH 10
W95L/N249Y
the mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD+ and NADH compared to the wild type enzyme, optimum pH is at about pH 8.6
D223G
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highly reduced activity compared to the wild-type enzyme
D223G/G225R
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nearly inactive mutant
D49N
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highly reduced activity compared to the wild-type enzyme
DELTAA200/A201L
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highly reduced activity compared to the wild-type enzyme
E68Q
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highly reduced activity compared to the wild-type enzyme
G204A
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nearly inactive mutant
G224I
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reduced activity compared to the wild-type enzyme
G225R
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reduced activity compared to the wild-type enzyme
H47R
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reduced activity compared to the wild-type enzyme
H51E
-
highly reduced activity compared to the wild-type enzyme
H51Q
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reduced activity compared to the wild-type enzyme
L203A
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reduced activity compared to the wild-type enzyme
L203A/T178S
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reduced activity compared to the wild-type enzyme
S110P/Y295C
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mutant is able to catalyze the NADH-dependent reduction of 5-hydroxymethylfurfural, an inhibitor of yeast fermentation, best activity among the mutants isolated
S198F
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highly reduced activity compared to the wild-type enzyme
S269I
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nearly inactive mutant
T48S
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reduced activity compared to the wild-type enzyme
T48S/T93A
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reduced activity compared to the wild-type enzyme
T48S/W57M/W93A
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reduced activity compared to the wild-type enzyme
W57L
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reduced activity compared to the wild-type enzyme
W57M
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slightly reduced activity compared to the wild-type enzyme
W93A
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reduced activity compared to the wild-type enzyme
Y295C
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mutant is able to catalyze the NADH-dependent reduction of 5-hydroxymethylfurfural, an inhibitor of yeast fermentation
S110P/Y295C
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mutant is able to catalyze the NADH-dependent reduction of 5-hydroxymethylfurfural, an inhibitor of yeast fermentation, best activity among the mutants isolated
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Y295C
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mutant is able to catalyze the NADH-dependent reduction of 5-hydroxymethylfurfural, an inhibitor of yeast fermentation
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I151V
polymorphism of the commercial yeast ADH (Saccharomyces pastorianus) compared to the laboratory strain Saccharomyces cerevisiae, in the V58T, Q127E, Q147E, and I151V substitutions, the removal of the methyl group has only weak effects on the structure of the neighboring residues
Q127E
polymorphism of the commercial yeast ADH (Saccharomyces pastorianus) compared to the laboratory strain Saccharomyces cerevisiae, in the V58T, Q127E, Q147E, and I151V substitutions, not readily distinguishable at the current resolutions, and the local structure is not significantly altered
Q147E
polymorphism of the commercial yeast ADH (Saccharomyces pastorianus) compared to the laboratory strain Saccharomyces cerevisiae, in the V58T, Q127E, Q147E, and I151V substitutions, not readily distinguishable at the current resolutions, and the local structure is not significantly altered
V58T
polymorphism of the commercial yeast ADH (Saccharomyces pastorianus) compared to the laboratory strain Saccharomyces cerevisiae, in the V58T, Q127E, Q147E, and I151V substitutions, not readily distinguishable at the current resolutions, and the local structure is not significantly altered
P704L/H734R
mutation leads to ethanol-tolerant phenotype. P704L may affect cofactor-binding directly, while mutation H734R is close to the active site iron atom. Mutant displays a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity
P704L/H734R
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mutation leads to ethanol-tolerant phenotype. P704L may affect cofactor-binding directly, while mutation H734R is close to the active site iron atom. Mutant displays a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity
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Y25A
kinetic parameters and temperature dependencies similar to wild-type
Y25A
mutation in the dimer-dimer interface, results in kinetic behavior similar to that of mutantion W87A
A25Y
mutation in the dimer-dimer interface, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature
A25Y
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mutation in the dimer-dimer interface, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature
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Y294F/W295A
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site-directed mutagenesis, compared to the wild-type enzyme, a shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol are observed
Y294F/W295A
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site-directed mutagenesis, mutant A2 variant
Y294F/W295A
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site-directed mutagenesis, compared to the wild-type enzyme, a shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol are observed
-
Y294F/W295A
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site-directed mutagenesis, mutant A2 variant
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N249Y
mutant exhibits increased catalytic activity compared to the wild type enzyme
N249Y
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mutant shows increased thermal stability and increased catalytic activity compared to the wild type enzyme
N249Y
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shows 7fold higher specific activity compared to the wild type enzyme, is active up to 95°C and more stable than the native enzyme, has acquired an improved resistance to proteolysis by thermolysin and shows a decreased activating effect by treatment with denaturants at moderate concentration
M294L
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7-10fold increase in reactivity, V/Km, with butanol, pentanol and hexanol
M294L
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increased activity compared to the wild-type enzyme
additional information
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biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE), process optimization, overview
additional information
biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE)
additional information
biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE)
additional information
generation of ADH1 gene disruption mutants
additional information
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generation of ADH1 gene disruption mutants
additional information
generation of enzyme overexpressing mutant strain 10-6C, which is a photosynthetic mutant impaired in CO2 assimilation because of a point mutation in the RBCL gene
additional information
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generation of enzyme overexpressing mutant strain 10-6C, which is a photosynthetic mutant impaired in CO2 assimilation because of a point mutation in the RBCL gene
additional information
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generation of enzyme overexpressing mutant strain 10-6C, which is a photosynthetic mutant impaired in CO2 assimilation because of a point mutation in the RBCL gene
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additional information
mutations Y25A (at the dimer interface) and V260A (at the cofactor-binding domain) exhibit opposing low-temperature effects on the hydride tunneling step. The distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process, and the double mutant (Y25A/V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. V260A displays a structural change in the active-site environment/solvation
additional information
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biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE)
additional information
biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE)
additional information
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biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE)
-
additional information
biotransformation studies with the recombinant enzyme expressed in Escherichia coli strain BL21(DE3) cells using the cell culture. Asymmetric biotransformation of (S)-ethyl-4-chloro-3-hydroxybutanoate ((S)-CHBE), process optimization, overview
additional information
generation of the ADH3 deletion mutant, complementation of the HpADH3 mutant by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium, while in a xylose medium, the ethanol production is dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain, phenotype analysis of DELTAHpADH3 and DELTAHpADH1/DELTAHpADH3 mutants, overview
additional information
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generation of the ADH3 deletion mutant, complementation of the HpADH3 mutant by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium, while in a xylose medium, the ethanol production is dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain, phenotype analysis of DELTAHpADH3 and DELTAHpADH1/DELTAHpADH3 mutants, overview
additional information
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generation of the ADH3 deletion mutant, complementation of the HpADH3 mutant by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium, while in a xylose medium, the ethanol production is dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain, phenotype analysis of DELTAHpADH3 and DELTAHpADH1/DELTAHpADH3 mutants, overview
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additional information
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expression of alcohol dehydrogenase domain alone, a minimal functional unit corresponds to amino acids 459-869
additional information
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expression of alcohol dehydrogenase domain alone, a minimal functional unit corresponds to amino acids 459-869
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additional information
replacement of three mobile loops positioned at the top of the canonical (alpha/beta)8-barrel structure by those from human aldose reductase. Replacement of Loops A and B is sufficient to impart from human aldose reductase activity into AdhD, and the resulting chimera retains the thermostability of the parent enzyme. No active chimeras are observed when the from human aldose reductase loops are grafted into a previously engineered cofactor specificity mutant of AdhD, which displays similar kinetics to from human aldose reductase with the model substrate DL-glyceraldehyde
additional information
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replacement of three mobile loops positioned at the top of the canonical (alpha/beta)8-barrel structure by those from human aldose reductase. Replacement of Loops A and B is sufficient to impart from human aldose reductase activity into AdhD, and the resulting chimera retains the thermostability of the parent enzyme. No active chimeras are observed when the from human aldose reductase loops are grafted into a previously engineered cofactor specificity mutant of AdhD, which displays similar kinetics to from human aldose reductase with the model substrate DL-glyceraldehyde
additional information
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insertion of an RTX domain from the adenylate cyclase of Bordetella pertussis into a loop near the catalytic active site of the thermostable alcohol dehydrogenase D from Pyrococcus furiosus. The resultant chimera, beta-AdhD, gains the calcium-binding ability of the beta-roll, retains the thermostable activity of AdhD, and exhibits reduced overall alcohol dehydrogenase activity. The addition of calcium to beta-AdhD preferentially inhibits NAD+-dependent activity in comparison to NADP+-dependent activity. Calcium is a competitive inhibitor of AdhD, and the addition of the RTX domain introduces calcium-dependent noncompetitive inhibition to beta-AdhD affecting NAD+-dependent activity. Thus, the insertion of an intrinsically disordered calcium-binding domain into a key loop in a cofactor-dependent enzyme results in an enzyme with tunable cofactor selectivity, reminiscent of a calcium-controlled cofactor selectivity rheostat switch
additional information
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generation of A2C2B1 variant, A2C3 variant, B1 variant, B1F4 variant, C1 variant, and C1B1 variant, crystal structure comparisons with the wild-type enzyme, overview
additional information
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generation of A2C2B1 variant, A2C3 variant, B1 variant, B1F4 variant, C1 variant, and C1B1 variant, crystal structure comparisons with the wild-type enzyme, overview
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additional information
C7IV28
expression of a mutant enzyme (with a glycine to aspartic acid mutation in the NADH binding site of the ADH domain of AdhE) in Pyrococcus furiosus from with the native aldehyde oxidoreductase (AOR) gene is deleted results in a reduced ethanol production to the background level
additional information
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expression of a mutant enzyme (with a glycine to aspartic acid mutation in the NADH binding site of the ADH domain of AdhE) in Pyrococcus furiosus from with the native aldehyde oxidoreductase (AOR) gene is deleted results in a reduced ethanol production to the background level
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