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The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dobson, R.C.; Valegard, K.; Gerrard, J.A.; J. Mol. Biol. 338, 329-339 (2004)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
overexpression of wild-type and mutant enzymes in strain XL1-Blue
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant wild-type and mutant enzymes, hanging or sitting drop vapour diffusion method, at 4°C and 21°C, 0.006 ml protein solutions: about 10 mg/ml protein, 1.8 M K2PO4, pH 10.0, N-octyl-beta-R-glucopyranoside 6% w/v, + 2 ml reservoir solution: 1.8 M K2PO4, pH 10.0, 3-4 days, X-ray diffraction structure determination and analysis at 2.35-2.5 A resolution
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
additional information
detailed structural properties of the mutant enzymes based on the crystal structure models
Escherichia coli
4.3.3.7
T44V
site-directed mutagenesis, reduced activity, structure is similar to the wild-type enzyme
Escherichia coli
4.3.3.7
Y107F
site-directed mutagenesis, reduced activity, structure is similar to the wild-type enzyme
Escherichia coli
4.3.3.7
Y133F
site-directed mutagenesis, reduced activity, structure is similar to the wild-type enzyme
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.08
-
pyruvate
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.09
-
(S)-aspartate 4-semialdehyde
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.11
-
(S)-aspartate 4-semialdehyde
wild-type enzyme, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.16
-
pyruvate
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.26
-
pyruvate
wild-type enzyme, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.58
-
(S)-aspartate 4-semialdehyde
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
2.7
-
(S)-aspartate 4-semialdehyde
mutant Y133F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
35
-
pyruvate
mutant Y133F, pH 8.0, 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
4.3.3.7
(S)-aspartate 4-semialdehyde + pyruvate
Escherichia coli
branch point reaction in the biosynthesis of lysine
dihydrodipicolinate + H2O
-
Escherichia coli
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Escherichia coli
P0A6L2
-
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
recombinant wild-type 5.8fold, recombinant mutant Y107F 17.6fold, recombinant mutant T44V 15.8fold, and recombinant mutant Y133F 18fold
Escherichia coli
Reaction
EC Number
Reaction
Commentary
Organism
4.3.3.7
pyruvate + L-aspartate-4-semialdehyde = (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
ping-pong kinetic and catalytic mechanism involves a catalytic triad which consists of Tyr133, Thr44, and Tyr107, overview
Escherichia coli
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
4.3.3.7
0.0034
-
purified mutant T44V
Escherichia coli
4.3.3.7
0.008
-
purified mutant Y133F
Escherichia coli
4.3.3.7
0.27
-
purified mutant Y107F
Escherichia coli
4.3.3.7
1.81
-
purified wild-type enzyme
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
-
652939
Escherichia coli
dihydrodipicolinate + H2O
-
652939
Escherichia coli
?
4.3.3.7
(S)-aspartate 4-semialdehyde + pyruvate
branch point reaction in the biosynthesis of lysine
652939
Escherichia coli
dihydrodipicolinate + H2O
-
652939
Escherichia coli
?
Subunits
EC Number
Subunits
Commentary
Organism
4.3.3.7
tetramer
homotetramer of (alpha/beta)8 barrels, each containing one active site, crystal structure
Escherichia coli
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.12
-
(S)-aspartate 4-semialdehyde
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.7
-
(S)-aspartate 4-semialdehyde
mutant Y133F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
10.8
-
(S)-aspartate 4-semialdehyde
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
124
-
(S)-aspartate 4-semialdehyde
wild-type enzyme, pH 8.0, 30°C
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
overexpression of wild-type and mutant enzymes in strain XL1-Blue
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant wild-type and mutant enzymes, hanging or sitting drop vapour diffusion method, at 4°C and 21°C, 0.006 ml protein solutions: about 10 mg/ml protein, 1.8 M K2PO4, pH 10.0, N-octyl-beta-R-glucopyranoside 6% w/v, + 2 ml reservoir solution: 1.8 M K2PO4, pH 10.0, 3-4 days, X-ray diffraction structure determination and analysis at 2.35-2.5 A resolution
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
additional information
detailed structural properties of the mutant enzymes based on the crystal structure models
Escherichia coli
4.3.3.7
T44V
site-directed mutagenesis, reduced activity, structure is similar to the wild-type enzyme
Escherichia coli
4.3.3.7
Y107F
site-directed mutagenesis, reduced activity, structure is similar to the wild-type enzyme
Escherichia coli
4.3.3.7
Y133F
site-directed mutagenesis, reduced activity, structure is similar 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
4.3.3.7
0.08
-
pyruvate
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.09
-
(S)-aspartate 4-semialdehyde
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.11
-
(S)-aspartate 4-semialdehyde
wild-type enzyme, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.16
-
pyruvate
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.26
-
pyruvate
wild-type enzyme, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.58
-
(S)-aspartate 4-semialdehyde
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
2.7
-
(S)-aspartate 4-semialdehyde
mutant Y133F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
35
-
pyruvate
mutant Y133F, pH 8.0, 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
4.3.3.7
(S)-aspartate 4-semialdehyde + pyruvate
Escherichia coli
branch point reaction in the biosynthesis of lysine
dihydrodipicolinate + H2O
-
Escherichia coli
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
recombinant wild-type 5.8fold, recombinant mutant Y107F 17.6fold, recombinant mutant T44V 15.8fold, and recombinant mutant Y133F 18fold
Escherichia coli
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
4.3.3.7
0.0034
-
purified mutant T44V
Escherichia coli
4.3.3.7
0.008
-
purified mutant Y133F
Escherichia coli
4.3.3.7
0.27
-
purified mutant Y107F
Escherichia coli
4.3.3.7
1.81
-
purified wild-type enzyme
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
-
652939
Escherichia coli
dihydrodipicolinate + H2O
-
652939
Escherichia coli
?
4.3.3.7
(S)-aspartate 4-semialdehyde + pyruvate
branch point reaction in the biosynthesis of lysine
652939
Escherichia coli
dihydrodipicolinate + H2O
-
652939
Escherichia coli
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.3.3.7
tetramer
homotetramer of (alpha/beta)8 barrels, each containing one active site, crystal structure
Escherichia coli
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.12
-
(S)-aspartate 4-semialdehyde
mutant T44V, pH 8.0, 30°C
Escherichia coli
4.3.3.7
0.7
-
(S)-aspartate 4-semialdehyde
mutant Y133F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
10.8
-
(S)-aspartate 4-semialdehyde
mutant Y107F, pH 8.0, 30°C
Escherichia coli
4.3.3.7
124
-
(S)-aspartate 4-semialdehyde
wild-type enzyme, pH 8.0, 30°C
Escherichia coli