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Critical catalytic functional groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase

Blanco, J.; Moore, R.A.; Faehnle, C.R.; Viola, R.E.; Acta Crystallogr. Sect. D 60, 1808-1815 (2004)

Data extracted from this reference:

Crystallization (Commentary)
EC Number
Crystallization
Organism
1.2.1.11
15 mg/ml purified recombinant wild-type enzyme in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, enzyme is free or complexed with substrates phosphate and/or asparate-beta-semialdehyde, hanging drop vapour diffusion method, 20C, against an equal volume of precipitant solution containing 24-28% PEG 3350, 0.2 M ammonium acetate, and 0.1 M Tris-HCl, pH 8.5, soaking of crystals before harvest in 100 mM phosphate and 2 mM aspartate-beta-semialdehyde, crystallization of mutant H277N in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, by addition of precipitant solution containing 5 mM NADP+ and 5 mM inhibitor S-methyl-L-cysteine sulfoxide, 22% PEG 3350, 0.2 M ammonium acetate and 0.1 M sodium cacodylate, pH 6.5, 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
C136S
site-directed mutagenesis, active site mutant is nearly inactive due to decrease in nuleophilicity, and also by a change in the orientation of the histidine imidazole ring
Haemophilus influenzae
1.2.1.11
H277N
site-directed mutagenesis, active site mutant shows 100fold decreased catalytic efficiency compared to the wild-type enzyme, shift in the position of the bound reaction intermediate
Haemophilus influenzae
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.2
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
0.5
-
L-aspartate-4-semialdehyde
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
1.1
-
NADP+
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
1.6
-
phosphate
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
2.7
-
phosphate
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
140
-
cacodylate
recombinant wild-type enzyme, pH 9.0, 30C
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
L-4-aspartyl phosphate + NADPH
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.2.1.11
Haemophilus influenzae
P44801
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, function of the catalytic nucleophile Cys136 and the acid-base catalytic His277, the latter is also stabilizing the hemithioacetal intermediate
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 + cacodylate + NADP+
10% of the activity with phosphate
654110
Haemophilus influenzae
L-4-aspartyl cacodylate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
reductive dephosphorylation in the aspartate biosynthetic pathway
654110
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
formation of an acyl-enzyme intermediate
654110
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
Temperature Optimum [C]
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.2.1.11
30
-
assay at
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
3.2
-
L-aspartate-4-semialdehyde
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
3.2
-
NADP+
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
3.2
-
phosphate
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
phosphate
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.2.1.11
9
-
assay at
Haemophilus influenzae
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Haemophilus influenzae
1.2.1.11
NADPH
-
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
15 mg/ml purified recombinant wild-type enzyme in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, enzyme is free or complexed with substrates phosphate and/or asparate-beta-semialdehyde, hanging drop vapour diffusion method, 20C, against an equal volume of precipitant solution containing 24-28% PEG 3350, 0.2 M ammonium acetate, and 0.1 M Tris-HCl, pH 8.5, soaking of crystals before harvest in 100 mM phosphate and 2 mM aspartate-beta-semialdehyde, crystallization of mutant H277N in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, by addition of precipitant solution containing 5 mM NADP+ and 5 mM inhibitor S-methyl-L-cysteine sulfoxide, 22% PEG 3350, 0.2 M ammonium acetate and 0.1 M sodium cacodylate, pH 6.5, 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
C136S
site-directed mutagenesis, active site mutant is nearly inactive due to decrease in nuleophilicity, and also by a change in the orientation of the histidine imidazole ring
Haemophilus influenzae
1.2.1.11
H277N
site-directed mutagenesis, active site mutant shows 100fold decreased catalytic efficiency compared to the wild-type enzyme, shift in the position of the bound reaction intermediate
Haemophilus influenzae
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.2
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
0.2
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
0.5
-
L-aspartate-4-semialdehyde
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
1.1
-
NADP+
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
1.6
-
phosphate
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
2.7
-
phosphate
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
140
-
cacodylate
recombinant wild-type enzyme, pH 9.0, 30C
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
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 + cacodylate + NADP+
10% of the activity with phosphate
654110
Haemophilus influenzae
L-4-aspartyl cacodylate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
reductive dephosphorylation in the aspartate biosynthetic pathway
654110
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
1.2.1.11
L-aspartate-4-semialdehyde + phosphate + NADP+
formation of an acyl-enzyme intermediate
654110
Haemophilus influenzae
L-4-aspartyl phosphate + NADPH
-
-
-
r
Temperature Optimum [C] (protein specific)
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.2.1.11
30
-
assay at
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
3.2
-
L-aspartate-4-semialdehyde
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
3.2
-
NADP+
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
3.2
-
phosphate
recombinant mutant H277N, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
L-aspartate-4-semialdehyde
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
NADP+
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
1.2.1.11
330
-
phosphate
recombinant wild-type enzyme, pH 9.0, 30C
Haemophilus influenzae
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.2.1.11
9
-
assay at
Haemophilus influenzae