BRENDA - Enzyme Database show

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production

Geng, F.; Chen, Z.; Zheng, P.; Sun, J.; Zeng, A.; Appl. Microbiol. Biotechnol. 97, 1963-1971 (2012)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
4.3.3.7
L-lysine
mutagenesis of the lysine binding sites of the Corynebacterium glutamicum enzyme according to the residues in the Escherichia coli enzyme does not conver the expected feedback inhibition but an activation of the nezyme by L-lysine
Corynebacterium glutamicum
Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)
Corynebacterium glutamicum
4.3.3.7
gene dapA, recombinant expression of lysine-insensitive mutants in Escherichia coli strain MG1655 with a yield improved by 46% compared to the wild-type enzyme
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
A49K
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
A49P
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme and is still sensitive to L-lysine
Escherichia coli
4.3.3.7
A49W
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
E84T
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme and is insensitive to L-lysine
Escherichia coli
4.3.3.7
H56K
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme and is insensitive to L-lysine
Escherichia coli
4.3.3.7
K68H
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
L51K
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
L51T
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme and is sill sensitive to L-lysine
Escherichia coli
4.3.3.7
additional information
mutagenesis of the lysine binding sites of the Corynebacterium glutamicum enzyme according to the residues in the Escherichia coli enzyme does not conver the expected feedback inhibition but an activation of the nezyme by L-lysine
Corynebacterium glutamicum
4.3.3.7
additional information
feedback inhibition of the Escherichia coli enzyme by lysine is successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of the Corynabacterium glutamicum enzyme
Escherichia coli
4.3.3.7
P61A/T63L
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
P61A/T63L/A65H
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T63L
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T92E
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T96E
site-directed mutagenesis, the mutant is not inhibited by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H
site-directed mutagenesis, the mutant is not inhibited by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/P61A/T63L
site-directed mutagenesis, the mutant is activated by L-lysine 3fold shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/P61A/T63L/A65H
site-directed mutagenesis the mutant is activated by L-lysine 5fold shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/T63L
site-directed mutagenesis, the mutant is activated by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
(S)-lysine
feedback inhibition, feedback inhibition of the Escherichia coli enzyme by lysine is successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of the Corynabacterium glutamicum enzyme
Escherichia coli
4.3.3.7
additional information
no feedback inhibition by L-lysine
Corynebacterium glutamicum
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.137
-
(S)-aspartate-4-semialdehyde
recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.248
-
(S)-aspartate-4-semialdehyde
recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.3
-
(S)-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.556
-
pyruvate
recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.64
-
pyruvate
recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.827
-
pyruvate
recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Escherichia coli
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Corynebacterium glutamicum
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Corynebacterium glutamicum ATCC 13032
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Escherichia coli MG1655
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Corynebacterium glutamicum
-
gene dapA
-
4.3.3.7
Corynebacterium glutamicum ATCC 13032
-
gene dapA
-
4.3.3.7
Escherichia coli
-
gene dapA
-
4.3.3.7
Escherichia coli MG1655
-
gene dapA
-
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
4.3.3.7
14.5
-
purified recombinant mutant L51K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
19.2
-
purified recombinant mutant A49K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
60.5
-
purified recombinant mutant L51T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
81.6
-
purified recombinant mutant A49W, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
454.2
-
purified recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
478.1
-
purified recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
500.8
-
purified recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
559.4
-
purified recombinant mutant A49P, pH 8.0, temperature not specified in the publication
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Escherichia coli
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Corynebacterium glutamicum
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Corynebacterium glutamicum ATCC 13032
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Escherichia coli MG1655
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
additional information
coupled assay with dihydrodipicolinate reductase
729062
Escherichia coli
?
-
-
-
-
4.3.3.7
additional information
coupled assay with dihydrodipicolinate reductase
729062
Escherichia coli MG1655
?
-
-
-
-
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Corynebacterium glutamicum
4.3.3.7
8
-
assay at
Escherichia coli
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
4.3.3.7
L-lysine
mutagenesis of the lysine binding sites of the Corynebacterium glutamicum enzyme according to the residues in the Escherichia coli enzyme does not conver the expected feedback inhibition but an activation of the nezyme by L-lysine
Corynebacterium glutamicum
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)
Corynebacterium glutamicum
4.3.3.7
gene dapA, recombinant expression of lysine-insensitive mutants in Escherichia coli strain MG1655 with a yield improved by 46% compared to the wild-type enzyme
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
A49K
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
A49P
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme and is still sensitive to L-lysine
Escherichia coli
4.3.3.7
A49W
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
E84T
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme and is insensitive to L-lysine
Escherichia coli
4.3.3.7
H56K
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme and is insensitive to L-lysine
Escherichia coli
4.3.3.7
K68H
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
L51K
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Escherichia coli
4.3.3.7
L51T
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme and is sill sensitive to L-lysine
Escherichia coli
4.3.3.7
additional information
mutagenesis of the lysine binding sites of the Corynebacterium glutamicum enzyme according to the residues in the Escherichia coli enzyme does not conver the expected feedback inhibition but an activation of the nezyme by L-lysine
Corynebacterium glutamicum
4.3.3.7
additional information
feedback inhibition of the Escherichia coli enzyme by lysine is successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of the Corynabacterium glutamicum enzyme
Escherichia coli
4.3.3.7
P61A/T63L
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
P61A/T63L/A65H
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T63L
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T92E
site-directed mutagenesis, the mutant is not inhibited by L-lysine
Corynebacterium glutamicum
4.3.3.7
T96E
site-directed mutagenesis, the mutant is not inhibited by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H
site-directed mutagenesis, the mutant is not inhibited by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/P61A/T63L
site-directed mutagenesis, the mutant is activated by L-lysine 3fold shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/P61A/T63L/A65H
site-directed mutagenesis the mutant is activated by L-lysine 5fold shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
4.3.3.7
T96E/K68H/T63L
site-directed mutagenesis, the mutant is activated by L-lysine and shows reduced activity compared to the wild-type enzyme
Corynebacterium glutamicum
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
(S)-lysine
feedback inhibition, feedback inhibition of the Escherichia coli enzyme by lysine is successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of the Corynabacterium glutamicum enzyme
Escherichia coli
4.3.3.7
additional information
no feedback inhibition by L-lysine
Corynebacterium glutamicum
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.137
-
(S)-aspartate-4-semialdehyde
recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.248
-
(S)-aspartate-4-semialdehyde
recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.3
-
(S)-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.556
-
pyruvate
recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.64
-
pyruvate
recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
0.827
-
pyruvate
recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
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
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Escherichia coli
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Corynebacterium glutamicum
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Corynebacterium glutamicum ATCC 13032
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Escherichia coli MG1655
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
4.3.3.7
14.5
-
purified recombinant mutant L51K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
19.2
-
purified recombinant mutant A49K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
60.5
-
purified recombinant mutant L51T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
81.6
-
purified recombinant mutant A49W, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
454.2
-
purified recombinant mutant H56K, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
478.1
-
purified recombinant mutant E84T, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
500.8
-
purified recombinant wild-type enzyme, pH 8.0, temperature not specified in the publication
Escherichia coli
4.3.3.7
559.4
-
purified recombinant mutant A49P, pH 8.0, temperature not specified in the publication
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Escherichia coli
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Corynebacterium glutamicum
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Corynebacterium glutamicum ATCC 13032
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
729062
Escherichia coli MG1655
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
additional information
coupled assay with dihydrodipicolinate reductase
729062
Escherichia coli
?
-
-
-
-
4.3.3.7
additional information
coupled assay with dihydrodipicolinate reductase
729062
Escherichia coli MG1655
?
-
-
-
-
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Corynebacterium glutamicum
4.3.3.7
8
-
assay at
Escherichia coli
General Information
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
feedback regulation of the enzyme is directly correlated to L-lysine production
Escherichia coli
4.3.3.7
additional information
structure comparison with the enzyme from Escherichia coli
Corynebacterium glutamicum
4.3.3.7
additional information
structure comparison with the enzyme from Corynebacterium glutamicum
Escherichia coli
General Information (protein specific)
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
feedback regulation of the enzyme is directly correlated to L-lysine production
Escherichia coli
4.3.3.7
additional information
structure comparison with the enzyme from Escherichia coli
Corynebacterium glutamicum
4.3.3.7
additional information
structure comparison with the enzyme from Corynebacterium glutamicum
Escherichia coli