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The role of substrate-binding groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase

Blanco, J.; Moore, R.A.; Faehnle, C.R.; Coe, D.M.; Viola, R.E.; Acta Crystallogr. Sect. D 60, 1388-1395 (2004)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.2.1.11
expression of wild-type and mutant enzymes
Haemophilus influenzae
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.2.1.11
10 mg/ml purified recombinant wild-type and mutant enzymes free or in complex with the substrates, protein in 10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM DTT, by hanging drop vapour diffusion, 20°C, mixed with equal volume of precipitant solution containing 22-24% PEG 3350, and 0.2 M ammonium acetate, crystals are soaked for 1 h in a solution containing 2 mM aspartate-beta-semialdehyde or 100 mM phosphate, 26% PEG 3350, 0.2 M ammonium acetate, 0.1 M Tris-HCl, pH 8.5, and 20% glycerol, X-ray diffraction structure determination and analysis at about 2.0 A resolution
Haemophilus influenzae
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.2.1.11
E243D
site-directed mutagenesis, unaltered Km for the substrates but 8fold increased Km for cofactor NADP+, active site structural alterations
Haemophilus influenzae
1.2.1.11
K246R
site-directed mutagenesis, mutation of a putative phosphate binding residue, unaltered substrate Km, active site structural alterations
Haemophilus influenzae
1.2.1.11
R103K
site-directed mutagenesis, adversely affected interaction between enzyme and phosphate, active site structural alterations
Haemophilus influenzae
1.2.1.11
R103L
site-directed mutagenesis, altered interaction between enzyme and phosphate, active site structural alterations
Haemophilus influenzae
1.2.1.11
R270K
site-directed mutagenesis, active site mutant, unaltered substrate Km, active site structural alterations
Haemophilus influenzae
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.03
-
L-aspartate-4-semialdehyde
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.1
-
L-aspartate-4-semialdehyde
recombinant mutant R103K and mutant K246L
Haemophilus influenzae
1.2.1.11
0.11
-
NADP+
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.17
-
NADP+
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
0.2
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme and mutant E243D
Haemophilus influenzae
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme
Haemophilus influenzae
1.2.1.11
0.4
-
L-aspartate-4-semialdehyde
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
0.6
-
NADP+
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
0.7
-
NADP+
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
1
-
phosphate
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
1.5
-
phosphate
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
1.6
-
NADP+
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
1.6
-
phosphate
recombinant wild-type enzyme
Haemophilus influenzae
1.2.1.11
1.9
-
phosphate
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
36.6
-
phosphate
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
240
-
phosphate
recombinant mutant R103L
Haemophilus influenzae
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+
Haemophilus influenzae
reductive dephosphorylation in the aspartate biosynthetic pathway, aspartate-beta-semialdehydr is the key intermediate in biosynthesis of diaminopimelic acid
L-4-aspartyl phosphate + NADPH
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.2.1.11
Haemophilus influenzae
-
gene asd
-
Reaction
EC Number
Reaction
Commentary
Organism
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+ = L-4-aspartyl phosphate + NADPH + H+
catalytic mechanism, role of substrate binding groups, residues Arg270, Glu243, Arg103, and Lys246 are involved
Haemophilus influenzae
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 + NADP+
reductive dephosphorylation in the aspartate biosynthetic pathway, aspartate-beta-semialdehydr is the key intermediate in biosynthesis of diaminopimelic acid
654103
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
formation of an acyl-enzyme intermediate
654103
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.2.1.11
additional information
-
stability of recombinant wild-type and mutant enzymes, oveview
Haemophilus influenzae
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.24
-
L-aspartate-4-semialdehyde
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.4
-
L-aspartate-4-semialdehyde
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
1.2
-
L-aspartate-4-semialdehyde
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
4
-
L-aspartate-4-semialdehyde
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
11
-
L-aspartate-4-semialdehyde
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
330
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme
Haemophilus influenzae
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Haemophilus influenzae
1.2.1.11
NADPH
-
Haemophilus influenzae
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
expression of wild-type and mutant enzymes
Haemophilus influenzae
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Haemophilus influenzae
1.2.1.11
NADPH
-
Haemophilus influenzae
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.2.1.11
10 mg/ml purified recombinant wild-type and mutant enzymes free or in complex with the substrates, protein in 10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM DTT, by hanging drop vapour diffusion, 20°C, mixed with equal volume of precipitant solution containing 22-24% PEG 3350, and 0.2 M ammonium acetate, crystals are soaked for 1 h in a solution containing 2 mM aspartate-beta-semialdehyde or 100 mM phosphate, 26% PEG 3350, 0.2 M ammonium acetate, 0.1 M Tris-HCl, pH 8.5, and 20% glycerol, X-ray diffraction structure determination and analysis at about 2.0 A resolution
Haemophilus influenzae
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.2.1.11
E243D
site-directed mutagenesis, unaltered Km for the substrates but 8fold increased Km for cofactor NADP+, active site structural alterations
Haemophilus influenzae
1.2.1.11
K246R
site-directed mutagenesis, mutation of a putative phosphate binding residue, unaltered substrate Km, active site structural alterations
Haemophilus influenzae
1.2.1.11
R103K
site-directed mutagenesis, adversely affected interaction between enzyme and phosphate, active site structural alterations
Haemophilus influenzae
1.2.1.11
R103L
site-directed mutagenesis, altered interaction between enzyme and phosphate, active site structural alterations
Haemophilus influenzae
1.2.1.11
R270K
site-directed mutagenesis, active site mutant, unaltered substrate Km, active site structural alterations
Haemophilus influenzae
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.03
-
L-aspartate-4-semialdehyde
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.1
-
L-aspartate-4-semialdehyde
recombinant mutant R103K and mutant K246L
Haemophilus influenzae
1.2.1.11
0.11
-
NADP+
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.17
-
NADP+
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
0.2
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme and mutant E243D
Haemophilus influenzae
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme
Haemophilus influenzae
1.2.1.11
0.4
-
L-aspartate-4-semialdehyde
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
0.6
-
NADP+
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
0.7
-
NADP+
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
1
-
phosphate
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
1.5
-
phosphate
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
1.6
-
NADP+
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
1.6
-
phosphate
recombinant wild-type enzyme
Haemophilus influenzae
1.2.1.11
1.9
-
phosphate
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
36.6
-
phosphate
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
240
-
phosphate
recombinant mutant R103L
Haemophilus influenzae
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+
Haemophilus influenzae
reductive dephosphorylation in the aspartate biosynthetic pathway, aspartate-beta-semialdehydr is the key intermediate in biosynthesis of diaminopimelic acid
L-4-aspartyl phosphate + NADPH
-
-
r
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 + NADP+
reductive dephosphorylation in the aspartate biosynthetic pathway, aspartate-beta-semialdehydr is the key intermediate in biosynthesis of diaminopimelic acid
654103
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
formation of an acyl-enzyme intermediate
654103
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.2.1.11
additional information
-
stability of recombinant wild-type and mutant enzymes, oveview
Haemophilus influenzae
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
0.24
-
L-aspartate-4-semialdehyde
recombinant mutant R103L
Haemophilus influenzae
1.2.1.11
0.4
-
L-aspartate-4-semialdehyde
recombinant mutant R270K
Haemophilus influenzae
1.2.1.11
1.2
-
L-aspartate-4-semialdehyde
recombinant mutant R103K
Haemophilus influenzae
1.2.1.11
4
-
L-aspartate-4-semialdehyde
recombinant mutant E243D
Haemophilus influenzae
1.2.1.11
11
-
L-aspartate-4-semialdehyde
recombinant mutant K246L
Haemophilus influenzae
1.2.1.11
330
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme
Haemophilus influenzae