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E131A
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the mutant shows a 2.3fold decrease in turnover number compared to the wild type enzyme
H186A
reduced activity compared to the wild type enzyme
H187A
reduced activity compared to the wild type enzyme
K260A
reduced activity compared to the wild type enzyme
M13F
mutant showing decrease in affinity for NADP+, but not NADPH
M13I
mutant showing decrease in affinity for NADP+, but not NADPH
R287A
reduced activity compared to the wild type enzyme
R446A
reduced activity compared to the wild type enzyme
S128A
reduced activity compared to the wild type enzyme
T262A
activity similar to the wild type enzyme
Y191A
reduced activity compared to the wild type enzyme
V244D/C257R
site-directed mutagenesis, unaltered reaction kinetics and increased stability, due introduction of 2 salt bridges by the double-mutation, compared to the wild-type enzyme
T35S
D4ZTT4
NADPH acts as a competitive inhibitor of NADP+ for the wild-type enzyme and as a non-competitive inhibitor of NADP+ for mutant enzyme T35S
T35S
D4ZTT4
wild-type enzyme shows higher affinity for NADP+ than mutant enzyme T35S
T35S
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NADPH acts as a competitive inhibitor of NADP+ for the wild-type enzyme and as a non-competitive inhibitor of NADP+ for mutant enzyme T35S
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T35S
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wild-type enzyme shows higher affinity for NADP+ than mutant enzyme T35S
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A30C/R31I/T32K
the mutant exhibits a 118.5fold reversal of coenzyme selectivity from NADP+ to NAD+
A30C/R31I/T32K
saturation mutagenesis, the mutant shows better kinetics and increased activity for NAD+ compared to the wild-type enzyme
A30D/R31I/T32I
the mutant exhibits a 4278fold reversal of coenzyme selectivity from NADP+ to NAD+
A30D/R31I/T32I
saturation mutagenesis, the mutant shows much better kinetics and strongly increased activity for NAD+ compared to the wild-type enzyme
A30E/R31I/T32D
the mutant exhibits a 1293fold reversal of coenzyme selectivity from NADP+ to NAD+
A30E/R31I/T32D
saturation mutagenesis, the mutant shows better kinetics and increased activity for NAD+ compared to the wild-type enzyme
R31I
the mutant exhibits a 6.9fold reversal of coenzyme selectivity from NADP+ to NAD+
R31I
saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme
R31I/T32G
the mutant exhibits a 22.1fold reversal of coenzyme selectivity from NADP+ to NAD+
R31I/T32G
saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme
R31T
the mutant exhibits a 3.5fold reversal of coenzyme selectivity from NADP+ to NAD+
R31T
saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme
M13C
mutant showing decrease in affinity for NADP+, but not NADPH
M13C
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the mutant shows a decrease in turnover number compared to the wild type enzyme
M13Q
mutant showing decrease in affinity for NADP+, but not NADPH
M13Q
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the mutant shows a 8.1fold decrease in turnover number compared to the wild type enzyme
M13V
mutant showing decrease in affinity for NADP+, but not NADPH
M13V
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the mutant shows a decrease in turnover number compared to the wild type enzyme
W104L
all kinetic parameters, especially the KM-value for 6-phospho-D-gluconate, are decreased in the mutant
W104L
all kinetic parameters, especially the KM-value for 6-phosphogluconate, are decreased in the mutant
S42T
amino acid substitution at position 42 from serine to threonine enhances the affinity for NADP+ and alters the mode of inhibition by NADPH from non-competitive (wild-type enzyme) to competitive (mutant enzyme S42T)
S42T
the amino acid substitution (S42T) enhances the affinity for NADP+ and alters the mode of inhibition by NADPH
N32D
the mutant exhibits an 460times higher Km value on NADP+ and a slightly decreased Km value of 4.0 mM on NAD+ compared with the wild type enzyme
N32D
site-directed mutagenesis, mutant N32D has a 460times higher Km value for NADP+ and a slightly decreased Km value for NAD+, but no significant difference of kcat for NADP+, compared to the wild-type enzyme
N32D/R33I/T34I
the mutant exhibits a far higher Km value of on NADP+ and a slightly decreased Km value on NAD+ compared with the wild type enzyme
N32D/R33I/T34I
site-directed mutagenesis, the triple mutant exhibits a far higher Km value for NADP+ and a slightly decreased Km value for NAD+, but no significant difference of kcat for NADP+, compared to the wild-type enzyme
N32D/R33L/T34S
the mutant has an increased Km value on NADP+ and NAD+ compared to the wild type enzyme
N32D/R33L/T34S
site-directed mutagenesis, the triple mutant has a increased Km value for NADP+ and NAD+, but no significant difference of kcat for NADP+, compared to the wild-type enzyme
N32E/R33I/T34I
the mutant exhibits an about 6400fold reversal of the coenzyme selectivity from NADP+ to NAD+ compared to the wild type enzyme
N32E/R33I/T34I
site-directed mutagenesis, the mutant shows almost 2fold declined Km and a 2fold increased kcat for NAD+ compared to wild-type, the catalytic efficiency kcat/Km towards NADP+ decreases, while the catalytic efficiency towards NAD+ increases
additional information
construction of a knockout mutant
additional information
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construction of a knockout mutant
additional information
high-throughput screening of the two-site saturation mutagenesis library A30/T32 in Escherichia coli TOP10 by using a pair of degenerate primers 30_32_NNK_F/30_32_NNK_R, enzyme mutant identification and selection by heat treatment, usage of the dual T7-tac promoter and a colorimetric assay, method optimization, overview
additional information
truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lose their 6PGDH activity
additional information
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truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lose their 6PGDH activity
additional information
construction and evaluation of enzyme mutants with increased activity for NAD* compared to the wild-type enzyme