Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes | Haemophilus influenzae |
Crystallization (Comment) | Organism |
---|---|
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 |
Protein Variants | Comment | Organism |
---|---|---|
E243D | site-directed mutagenesis, unaltered Km for the substrates but 8fold increased Km for cofactor NADP+, active site structural alterations | Haemophilus influenzae |
K246R | site-directed mutagenesis, mutation of a putative phosphate binding residue, unaltered substrate Km, active site structural alterations | Haemophilus influenzae |
R103K | site-directed mutagenesis, adversely affected interaction between enzyme and phosphate, active site structural alterations | Haemophilus influenzae |
R103L | site-directed mutagenesis, altered interaction between enzyme and phosphate, active site structural alterations | Haemophilus influenzae |
R270K | site-directed mutagenesis, active site mutant, unaltered substrate Km, active site structural alterations | Haemophilus influenzae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.03 | - |
L-aspartate-4-semialdehyde | recombinant mutant R103L | Haemophilus influenzae | |
0.1 | - |
L-aspartate-4-semialdehyde | recombinant mutant R103K and mutant K246L | Haemophilus influenzae | |
0.11 | - |
NADP+ | recombinant mutant R103L | Haemophilus influenzae | |
0.17 | - |
NADP+ | recombinant mutant R270K | Haemophilus influenzae | |
0.2 | - |
NADP+ | recombinant wild-type enzyme | Haemophilus influenzae | |
0.2 | - |
L-aspartate-4-semialdehyde | recombinant wild-type enzyme and mutant E243D | Haemophilus influenzae | |
0.4 | - |
L-aspartate-4-semialdehyde | recombinant mutant R270K | Haemophilus influenzae | |
0.6 | - |
NADP+ | recombinant mutant K246L | Haemophilus influenzae | |
0.7 | - |
NADP+ | recombinant mutant R103K | Haemophilus influenzae | |
1 | - |
phosphate | recombinant mutant K246L | Haemophilus influenzae | |
1.5 | - |
phosphate | recombinant mutant E243D | Haemophilus influenzae | |
1.6 | - |
phosphate | recombinant wild-type enzyme | Haemophilus influenzae | |
1.6 | - |
NADP+ | recombinant mutant E243D | Haemophilus influenzae | |
1.9 | - |
phosphate | recombinant mutant R270K | Haemophilus influenzae | |
36.6 | - |
phosphate | recombinant mutant R103K | Haemophilus influenzae | |
240 | - |
phosphate | recombinant mutant R103L | Haemophilus influenzae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Haemophilus influenzae | - |
gene asd | - |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Haemophilus influenzae | L-4-aspartyl phosphate + NADPH | - |
r | |
L-aspartate-4-semialdehyde + phosphate + NADP+ | formation of an acyl-enzyme intermediate | Haemophilus influenzae | L-4-aspartyl phosphate + NADPH | - |
r |
Synonyms | Comment | Organism |
---|---|---|
ASADH | - |
Haemophilus influenzae |
aspartate-beta-semialdehyde dehydrogenase | - |
Haemophilus influenzae |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
stability of recombinant wild-type and mutant enzymes, oveview | Haemophilus influenzae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.24 | - |
L-aspartate-4-semialdehyde | recombinant mutant R103L | Haemophilus influenzae | |
0.4 | - |
L-aspartate-4-semialdehyde | recombinant mutant R270K | Haemophilus influenzae | |
1.2 | - |
L-aspartate-4-semialdehyde | recombinant mutant R103K | Haemophilus influenzae | |
4 | - |
L-aspartate-4-semialdehyde | recombinant mutant E243D | Haemophilus influenzae | |
11 | - |
L-aspartate-4-semialdehyde | recombinant mutant K246L | Haemophilus influenzae | |
330 | - |
L-aspartate-4-semialdehyde | recombinant wild-type enzyme | Haemophilus influenzae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Haemophilus influenzae | |
NADPH | - |
Haemophilus influenzae |