Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
E121A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate, but is active with N-acetyl- and N-formyl-L-aspartate | Escherichia coli |
E121D | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate | Escherichia coli |
E121K | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate | Escherichia coli |
E121Q | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate | Escherichia coli |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
D-Aspartate | - |
Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.038 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.048 | - |
L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.61 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121Q | Escherichia coli | |
2.9 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121K | Escherichia coli | |
3.2 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
3.7 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
4.7 | - |
3-hydroxy-erythro-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
17 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
17.5 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
26.8 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121D | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-aspartate + fumarate | Escherichia coli | - |
iminosuccinate + succinate | - |
? | |
L-aspartate + O2 | Escherichia coli | - |
iminosuccinate + H2O2 | - |
? | |
additional information | Escherichia coli | the enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. No activity with 3-hydroxy-threo-L-aspartate | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-hydroxy-erythro-L-aspartate + O2 | - |
Escherichia coli | 2-amino-3-hydroxy-2-butenedioic acid + H2O2 | - |
? | |
L-aspartate + fumarate | - |
Escherichia coli | iminosuccinate + succinate | - |
? | |
L-aspartate + O2 | - |
Escherichia coli | iminosuccinate + H2O2 | - |
? | |
additional information | the enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. No activity with 3-hydroxy-threo-L-aspartate | Escherichia coli | ? | - |
? | |
additional information | the enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. Catalytic role of the active site residue E121, substrate specificity of wild-type and mutant enzymes, molecular docking studies, role of R290, overview. E121 interacts favourably with the charged amino group of the substrate and different ligands might assume different orientations in the active site of the enzyme, binding modes for L-aspartate, overview | Escherichia coli | ? | - |
? | |
N-acetyl-L-aspartate + O2 | - |
Escherichia coli | ? + H2O2 | - |
? | |
N-formyl-L-aspartate + O2 | - |
Escherichia coli | ? + H2O2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | three dimensional structure of holo-wild-type-LASPO, modelling, overview | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
LASPO | - |
Escherichia coli |
More | LASPO belongs to the succinate dehydrogenase/fumarate reductase family | Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0013 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.0035 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.0055 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.0077 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.173 | - |
3-hydroxy-erythro-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.173 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.202 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121Q | Escherichia coli | |
0.215 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121D | Escherichia coli | |
0.223 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121K | Escherichia coli | |
0.267 | - |
L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | E121 belongs to a polypeptide stretch, residues 119-127, involved in the interdomain contact between the FAD and the capping domains and is hydrogen bound to G51, itself belonging to a loop of residues 49-57 playing a key role in coenzyme binding | Escherichia coli |
General Information | Comment | Organism |
---|---|---|
additional information | LASPO displays strong primary and tertiary structure similarity with the flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate 121, which is substituted by apolar amino acids in the other enzymes | Escherichia coli |
physiological function | LASPO is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD+. The enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00007 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.00021 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.0017 | - |
N-acetyl-L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
0.00208 | - |
N-formyl-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.008 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121D | Escherichia coli | |
0.0368 | - |
3-hydroxy-erythro-L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli | |
0.077 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121K | Escherichia coli | |
0.331 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121Q | Escherichia coli | |
4.55 | - |
L-aspartate | pH 8.0, 25°C, recombinant mutant E121A | Escherichia coli | |
5.56 | - |
L-aspartate | pH 8.0, 25°C, recombinant wild-type enzyme | Escherichia coli |