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C201M/C332Y/K344D/Y345I/V351A/Y391K/R395S
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converts the cofactor specificity from 7000-fold preference of NADP+ to a 200-fold preference of NAD+
alphaY126E
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site-directed mutagenesis of subunit alpha, almost inactive mutant, shows low activity at pH 6.1 instead of pH 7.2, Km,Mn2+ is 30fold higher in the alphaY126E mutant as compared with the wild-type. Km,NAD+ for the alphaY126E mutant is 29fold higher than that of the wild-type. The Vmax of the wild-type at pH 6.1 is 0.0144 mmol/min/mg, whereas that for the alphaY126E mutant is only 0.00103 mmol/min/mg, suggesting a critical role for the residue in enzyme activity
alphaY126F
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site-directed mutagenesis of subunit alpha, inactive mutant
alphaY126S
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site-directed mutagenesis of subunit alpha, inactive mutant
betaY137E
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site-directed mutagenesis of subunit beta, the mutant shows reduced activity compared to the wild-type enzyme
betaY137F
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site-directed mutagenesis of subunit beta, the mutant shows reduced activity compared to the wild-type enzyme
betaY137S
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site-directed mutagenesis of subunit beta, the mutant shows reduced activity compared to the wild-type enzyme
D181N
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mutation in the alpha-subunit exhibits a 2000fold decrease in Vmax, with increases of 15fold in the Kms for Mn2+ and NAD+ and a much smaller change in the Km for isocitrate. Mutant enzyme fails to respond to ADP by lowering the Km for isocitrate, although it binds to ADP
D190N
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mutation in the gamma-subunit results in 4-5fold decrease in Vmax as compared to wild-type enzyme. The Km-values for NAD+ and for Mn2+ of the mutant enzyme are 19 and 72 times, respectively, that of the wild-type enzyme with a much smaller effect on the Km for isocitrate. Mutant enzyme fails to respond to ADP by lowering the Km for isocitrate, although it binds to ADP
D192N
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mutation in the beta-subunit results in 4-5fold decrease in Vmax as compared to wild-type enzyme. The Km-value for NAD+ of the mutant enzyme is 9times that of the normal enzyme with little or no effect on the affinity for Mn2+ or isocitrate. Mutant enzyme fails to respond to ADP by lowering the Km for isocitrate, although it binds to ADP
gammaY135F
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site-directed mutagenesis of subunit gamma, inactive mutant
L98P
the mutation is associated with retinitis pigmentosa
R132C
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naturally occuring IDH1 mutation
R132G
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naturally occuring IDH1 mutation
R132H
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naturally occuring IDH1 mutation
R132L
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naturally occuring IDH1 mutation
R132S
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naturally occuring IDH1 mutation
R132V
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naturally occuring IDH1 mutation
R88Q
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site-directed mutagenesis, residue of the alpha-subunit, inactive mutant
R97Q
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site-directed mutagenesis, residue of the additional mutant gamma-subunit, highly reduced activity compared to the wild-type enzyme
R99Q
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site-directed mutagenesis, residue of the beta-subunit, reduced activity compared to the wild-type enzyme
D326R/M327H
site-directed mutagenesis, the coenzyme specificity of the mutant CaIDH Asp326/Met327 is completely reversed from NAD+ to NADP+
D326X/M327X
site-directed mutagenesis, the coenzyme specificity of the mutant OlIDH R326H327 is completely reversed from NAD+ to NADP+
D326R/M327H
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site-directed mutagenesis, the coenzyme specificity of the mutant CaIDH Asp326/Met327 is completely reversed from NAD+ to NADP+
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D326X/M327X
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site-directed mutagenesis, the coenzyme specificity of the mutant OlIDH R326H327 is completely reversed from NAD+ to NADP+
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D344R
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site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, and the mutant displays a 15fold preference for NADP+ over NAD+ compared to the wild-type enzyme
D344R/M345H
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site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, the mutant displays a 72fold preference for NADP+ over NAD+ compared to the wild-type enzyme
D459A/N461A/D463A/F465A
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the mutant maintains comparable specific activity to that of wild type enzyme
IDH1-EF
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the mutant without the EF-hand domain (deleted Gln424-Val495 in a total of 72 residues) completely lost its catalytic activity
D459A/N461A/D463A/F465A
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the mutant maintains comparable specific activity to that of wild type enzyme
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IDH1-EF
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the mutant without the EF-hand domain (deleted Gln424-Val495 in a total of 72 residues) completely lost its catalytic activity
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D328K
the mutant shows dual coenzyme specificity
D328K/I329Y
introduction of the double mutation shifts the cofactor preference from NAD+ to NADP+
A108R/F136Y/T241D/N245D
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site-directed mutagenesis, residues of subunit IDH1 mutant is unable to bind AMP, ligand-binding analysis
A108R/F136Y/T241D/N245D/R114A/Y142F/D248T/D252N
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site-directed mutagenesis, residues of subunit IDH1 are A108R, F136Y, T241D, and N245D, residues of subunit IDH2 are R114A, Y142F, D248T, and D252N, mutant is unable to bind AMP, ligand-binding analysis
C56S/C150S/C242S
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site-directed mutagenesis, IDH1/IDH2C150S octameric enzyme
D279A/D280A
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site-directed mutagenesis, mutation of a IDH1 residue, reduced AMP binding, ligand-binding analysis
D286A/I287A
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site-directed mutagenesis, mutation of a IDH2 residue, highly reduced NAD+ binding, ligand-binding analysis
H281A
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site-directed mutagenesis, mutation of a IDH2 residue, mutant cannot bind NAD+, ligand-binding analysis
I221A
residue changes in IDH2 subunits
I221A/V225A/V229A
mutant enzyme IDH1-IDH2(I221A/V225A/V229A) shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. Mutant enzyme IDH1-IDH2 (I221A/V225A/V229A) exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
R114A/Y142F/D248T/D252N
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site-directed mutagenesis, residues of subunit subunit IDH2, ligand-binding analysis
R274A
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site-directed mutagenesis, mutation of a IDH1 residue, reduced AMP binding, ligand-binding analysis
S220A
residue changes in IDH1 subunits
S92A/S98A
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site-directed mutagenesis, residue of subunit IDH1 is S92, residue of subunit IDH2 is S98, ligand-binding analysis
V214A
residue changes in IDH1 subunits
V216A/S220A/V224A
IDH1(V216A/S220A/V224A)IDH2 mutant enzyme shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. IDH1(V216A/S220A/V224A)IDH2 mutant enzyme exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
V216A/S220A/V224A/I221A/V225A/V229A
in the absence of AMP, the IDH1(V216A/S220A/V224A)-IDH2(I221A/V225A/V229A) mutant enzyme demonstrates an about 30fold decrease in apparent Vmax relative to wild-type and a loss of cooperativity with respect to isocitrate and, in the presence of AMP, the apparent affinity of the enzyme for isocitrate is 35fold lower than that of the wild-type enzyme. This mutant enzyme also exhibits a significant decrease in apparent affinity for NAD+ (about 30fold in the absence of AMP and about 10fold in the presence of AMP relative to wild-type). The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme demonstrates decreases in apparent affnity for isocitrate of about 12fold in the absence of AMP and of about 26fold in the presence of AMP relative to wild-type. For this mutant enzyme, with respect to isocitrate, allosteric activation by AMP and cooperativity in the absence of AMP are no longer apparen. The mutant enzyme is not fully functional in vivo
V224A
residue changes in IDH1 subunits
V225A
residue changes in IDH2 subunits
V229A
residue changes in IDH2 subunits
C150S
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insensitive to treatment with diamide, the presence of the IDH2 Cys-150 residue (and perhaps the potential for disulfide bond formation) may contribute to cooperativity and may also limit maximal IDH activity
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C56S/C242S
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shows activity similar to wild type enzyme and is sensitive to treatment with diamide
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I221A
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residue changes in IDH2 subunits
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K171L
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mutant IDH1K171L
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S220A
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residue changes in IDH1 subunits
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V214A
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residue changes in IDH1 subunits
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V224A
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residue changes in IDH1 subunits
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V225A
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residue changes in IDH2 subunits
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S102A
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site-directed mutagenesis, the mutant shows decreased affinity for isocitrate and 3.3% of wild-type activity
S102G
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site-directed mutagenesis, the mutant shows decreased affinity for isocitrate and 2.8% of wild-type activity
S102T
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site-directed mutagenesis, the mutant shows decreased affinity for isocitrate and 16% of wild-type activity
S102Y
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site-directed mutagenesis, the mutant shows decreased affinity for isocitrate and 1.1% of wild-type activity
D268K
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the coenzyme specificity of the mutant is switched from NAD+ to NADP+
D268K/I269Y
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the coenzyme specificity of the mutant is switched from NAD+ to NADP+
D268K/I269Y/A275V
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the coenzyme specificity of the mutant is switched from NAD+ to NADP+
L580H/L591R/A640R
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the mutant has a 21fold lower Km value for NADP+ when compared to the original enzyme. The turnover rate (kcat) towards isocitrate of the mutant is reduced by more than 50% as compared to the wild type enzyme and even though the mutant is capable of using NADP+ for catalysis, ist catalytic efficiency (kcat/Km) for isocitrate is relatively low, being half of that for the wild type enzyme as using NAD+
L580H/L591R/A640R
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the mutant has a 21fold lower Km value for NADP+ when compared to the original enzyme. The turnover rate (kcat) towards isocitrate of the mutant is reduced by more than 50% as compared to the wild type enzyme and even though the mutant is capable of using NADP+ for catalysis, ist catalytic efficiency (kcat/Km) for isocitrate is relatively low, being half of that for the wild type enzyme as using NAD+
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L584H/D595R
site-directed mutagenesis, the coenzyme specificity of the mutant CaIDH H584/R595 is completely reversed from NAD+ to NADP+
L584H/D595R
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site-directed mutagenesis, the coenzyme specificity of the mutant CaIDH H584/R595 is completely reversed from NAD+ to NADP+
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D487R/L488H
site-directed mutagenesis, the coenzyme specificity of a the ClIDH mutant is altered compared to the wild-type enzyme, and the preference of the mutant for NADP+ is approximately 24fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup
D487R/L488H
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site-directed mutagenesis, the coenzyme specificity of a the ClIDH mutant is altered compared to the wild-type enzyme, and the preference of the mutant for NADP+ is approximately 24fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup
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D206N
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alpha-subunit mutant
D206N
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specific activity of the alpha-subunit mutant enzyme is 1.3fold higher than specific activity of wild-type enzyme
D215N
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gamma-subunit mutant
D215N
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specific activity of the gamma-subunit mutant enzyme is 17% of specific activity of wild-type enzyme. 100fold increase in KM-value for Mn2+. Km-value for isocitrate is elevated
D217N
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beta-subunit mutant
D217N
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specific activity of the beta-subunit mutant enzyme is 33% of specific activity of wild-type enzyme. 16fold increase in KM-vlaue for NAD+
D230C
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alpha-subunit mutant
D230C
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specific activity of the alpha-subunit mutant enzyme is 6% of specific activity of wild-type enzyme. 32fold increase in KM-value for Mn2+. Km-value for isocitrate is elevated. 16fold increase in KM-vlaue for NAD+
D230N
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alpha-subunit mutant
D230N
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mutation in alpha-subunit results in complete loss of activity
D234C
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alpha-subunit mutant
D234C
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specific activity of the alpha-subunit mutant enzyme is 1% of specific activity of wild-type enzyme
D234N
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alpha-subunit mutant
D234N
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mutation in alpha-subunit results in complete loss of activity
C150S
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insensitive to treatment with diamide, the presence of the IDH2 Cys-150 residue (and perhaps the potential for disulfide bond formation) may contribute to cooperativity and may also limit maximal IDH activity
C150S
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an octameric IDH1/IDH2C150S mutant enzyme, that shows unaltered activity and similar kinetics compared to the wild-type enzyme
C150S
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site-directed mutagenesis of subunit IDH2, disulfide bridge formation by C150 is abolished
C150S
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site-directed mutagenesis, IDH1/IDH2C150S octameric enzyme
C56S/C242S
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shows activity similar to wild type enzyme and is sensitive to treatment with diamide
C56S/C242S
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an octameric IDH1/IDH2C56S/C242S mutant enzyme, the mutant enzyme shows a reduction in Vmax relative to that of the wild-type enzyme of about 50%, although about 30% activity is restored in the presence of dithiothreitol
C56S/C242S
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site-directed mutagenesis of subunit IDH2, the mutation does not affect disulfide formation in the enzyme
C56S/C242S
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site-directed mutagenesis, IDH1/IDH2C150S octameric enzyme
D279A
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IDH1D279A
D279A
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site-directed mutagenesis of subunit IDH1, the mutation results in a loss of activation by AMP
D286A
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IDH2D286A
D286A
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site-directed mutagenesis of subunit IDH2, the mutation results in a dramatic reduction in Vmax primarily due to a 70fold increase in the S0.5 value for NAD+
G15D
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a tetrameric IDH1G15D/IDH2 mutant enzyme, that shows reduced activity and altered kinetics compared to the wild-type enzyme
G15D
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site-directed mutagenesis of subunit IDH1, the tetrameric IDH1G15D/IDH2 enzyme exhibits half-site binding (two sites) for isocitrate in the absence of DTT and full-site binding (four sites) in the presence of DTT
I280A
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IDH1I280A
I280A
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site-directed mutagenesis of subunit IDH1, the mutation results in a loss of activation by AMP
I287A
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IDH1I287A
I287A
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site-directed mutagenesis of subunit IDH2, the mutation results in a dramatic reduction in Vmax primarily due to a 70fold increase in the S0.5 value for NAD+
S92A
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site-directed mutagenesis, residue of subunit IDH1, reduction of isocitrate substrate binding sites by half, detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP, ligand-binding analysis
S98A
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site-directed mutagenesis, residue of subunit IDH2, reduction of isocitrate substrate binding sites by half, detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP, ligand-binding analysis
additional information
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construction of a knockout mutant of subunit IDH-II, disruption by dissociation insertion element, mutant strain shows normal growth and development, but reduced enzyme activity in mitochondria compared to the wild-type plants, no altered phenotype of the mutant plants, but slightly reduced growth
additional information
characterization of three IDH mutants corresponding to an insertion into a different IDH gene (At5g03290, idhv. At4g35260, idhi. At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively
additional information
characterization of three IDH mutants corresponding to an insertion into a different IDH gene (At5g03290, idhv. At4g35260, idhi. At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively
additional information
characterization of three IDH mutants corresponding to an insertion into a different IDH gene (At5g03290, idhv. At4g35260, idhi. At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively
additional information
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construction of a knockout mutant of subunit IDH-II, disruption by dissociation insertion element, mutant strain shows normal growth and development, but reduced enzyme activity in mitochondria compared to the wild-type plants, no altered phenotype of the mutant plants, but slightly reduced growth
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additional information
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mutational analysis of IDH1 codon 132 in 1185 cancer samples, overview
additional information
enzyme overexpression, or enzyme downregulation by siRNA
additional information
NAD-IDHalpha knockdown by shRNA
additional information
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NAD-IDHalpha knockdown by shRNA
additional information
enzyme overexpression, or enzyme downregulation by siRNA
additional information
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the enzyme's coenzyme specificity can be completely converted from NAD+ (ancient trait) to NADP+ (adaptive trait) by rational mutagenesis based on the evolutionary trace. Residues D344 and M345 are the determinants of NAD+ specificity
additional information
enzyme overexpression, or enzyme downregulation by siRNA
additional information
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transgenic tomato plants expressing the IDH gene in antisense orientation display a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield are, however, reduced