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Mutagenesis of key residues in the binding center of L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli enhances utilization of the cofactor NAD(H)

Xu, X.; Chen, J.; Wang, Q.; Duan, C.; Li, Y.; Wang, R.; Yang, S.; ChemBioChem 17, 56-64 (2016)

Data extracted from this reference:

Application
EC Number
Application
Commentary
Organism
1.2.1.11
biotechnology
the modofied enzyme with altered substrate specificity using NAD(H) is preferred in biotechnological production of amino acids due to lower costs and higher stability
Escherichia coli
1.2.1.11
synthesis
the modofied enzyme with altered substrate specificity using NAD(H) is preferred in biotechnological production of amino acids due to lower costs and higher stability
Escherichia coli
Cloned(Commentary)
EC Number
Commentary
Organism
1.2.1.11
gene asd, construction of a genetic ecASADH library by saturation mutagenesis, recombinant expression of His-tagged wild-type and selected mutants in Escherichia coli strain BL21(DE3)
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.2.1.11
A163S
site-directed mutagenesis, the mutant shows almost unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
H171
site-directed mutagenesis, the mutant shows almost unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
L351V
site-directed mutagenesis, the mutant shows unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
Q350N
site-directed mutagenesis, the mutant shows 44fold increased activity with NAD+ compared to the wild-type enzyme and can also also utilize NADH efficiently. Unlike the wild-type enzyme, mutants Q350N and Q350N/H171A are able to synthesize L-homoserine from aspartate efficiently with NADH as a cofactor
Escherichia coli
1.2.1.11
Q350N/H171A Q350N
site-directed mutagenesis, the mutant shows 66fold increased activity with NAD+ compared to the wild-type enzyme and can also utilize NADH efficiently. Unlike the wild-type enzyme, mutants Q350N and Q350N/H171A are able to synthesize L-homoserine from aspartate efficiently with NADH as a cofactor
Escherichia coli
1.2.1.11
S138Q
site-directed mutagenesis, the mutant shows unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.0026
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
0.0057
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
2.2
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
2.5
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
11.4
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
Escherichia coli
-
L-4-aspartyl phosphate + NADPH + H+
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.2.1.11
Escherichia coli
P0A9Q9
MG1655
-
Purification (Commentary)
EC Number
Commentary
Organism
1.2.1.11
recombinant His-tagged wild-type and mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NAD+
-
742287
Escherichia coli
L-4-aspartyl phosphate + NADH + H+
-
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
-
742287
Escherichia coli
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.2.1.11
30
-
assay at
Escherichia coli
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.2.1.11
9.5
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
13.4
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
53.4
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
86.2
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
115.2
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
258
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.2.1.11
9
-
assay at
Escherichia coli
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
additional information
cofactor modes of wild-type and mutant enzymes determined by molecular modeling
Escherichia coli
1.2.1.11
NAD+
very low activity with the wild-type enzyme, but 44fold and 66fold higher activity with enzyme mutants Q350N and Q350N/H171A, respectively, compared to the wild-type
Escherichia coli
1.2.1.11
NADH
-
Escherichia coli
1.2.1.11
NADP+
highly preferred by the wild-type enzyme
Escherichia coli
1.2.1.11
NADPH
-
Escherichia coli
Application (protein specific)
EC Number
Application
Commentary
Organism
1.2.1.11
biotechnology
the modofied enzyme with altered substrate specificity using NAD(H) is preferred in biotechnological production of amino acids due to lower costs and higher stability
Escherichia coli
1.2.1.11
synthesis
the modofied enzyme with altered substrate specificity using NAD(H) is preferred in biotechnological production of amino acids due to lower costs and higher stability
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
gene asd, construction of a genetic ecASADH library by saturation mutagenesis, recombinant expression of His-tagged wild-type and selected mutants in Escherichia coli strain BL21(DE3)
Escherichia coli
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
additional information
cofactor modes of wild-type and mutant enzymes determined by molecular modeling
Escherichia coli
1.2.1.11
NAD+
very low activity with the wild-type enzyme, but 44fold and 66fold higher activity with enzyme mutants Q350N and Q350N/H171A, respectively, compared to the wild-type
Escherichia coli
1.2.1.11
NADH
-
Escherichia coli
1.2.1.11
NADP+
highly preferred by the wild-type enzyme
Escherichia coli
1.2.1.11
NADPH
-
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.2.1.11
A163S
site-directed mutagenesis, the mutant shows almost unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
H171
site-directed mutagenesis, the mutant shows almost unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
L351V
site-directed mutagenesis, the mutant shows unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
1.2.1.11
Q350N
site-directed mutagenesis, the mutant shows 44fold increased activity with NAD+ compared to the wild-type enzyme and can also also utilize NADH efficiently. Unlike the wild-type enzyme, mutants Q350N and Q350N/H171A are able to synthesize L-homoserine from aspartate efficiently with NADH as a cofactor
Escherichia coli
1.2.1.11
Q350N/H171A Q350N
site-directed mutagenesis, the mutant shows 66fold increased activity with NAD+ compared to the wild-type enzyme and can also utilize NADH efficiently. Unlike the wild-type enzyme, mutants Q350N and Q350N/H171A are able to synthesize L-homoserine from aspartate efficiently with NADH as a cofactor
Escherichia coli
1.2.1.11
S138Q
site-directed mutagenesis, the mutant shows unaltered cofactor specificity compared to the wild-type enzyme
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.0026
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
0.0057
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
2.2
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
2.5
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
11.4
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
Escherichia coli
-
L-4-aspartyl phosphate + NADPH + H+
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
recombinant His-tagged wild-type and mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NAD+
-
742287
Escherichia coli
L-4-aspartyl phosphate + NADH + H+
-
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
-
742287
Escherichia coli
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.2.1.11
30
-
assay at
Escherichia coli
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.2.1.11
9.5
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
13.4
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
53.4
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
86.2
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
115.2
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
258
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.2.1.11
9
-
assay at
Escherichia coli
General Information
EC Number
General Information
Commentary
Organism
1.2.1.11
metabolism
the enzyme has a rate-limiting key function in the biosynthesis of amino acids L-threonine, L-lysine, and L-isoleucine from L-aspartate via L-homoserine
Escherichia coli
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.2.1.11
metabolism
the enzyme has a rate-limiting key function in the biosynthesis of amino acids L-threonine, L-lysine, and L-isoleucine from L-aspartate via L-homoserine
Escherichia coli
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.8
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
35.1
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
53.3
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
1124.9
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
5135.9
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
9612.8
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.8
-
NAD+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
35.1
-
NAD+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli
1.2.1.11
53.3
-
NAD+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
1124.9
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30°C
Escherichia coli
1.2.1.11
5135.9
-
NADP+
recombinant mutant Q350N/H171A, pH 9.0, 30°C
Escherichia coli
1.2.1.11
9612.8
-
NADP+
recombinant mutant Q350N, pH 9.0, 30°C
Escherichia coli