Cloned (Comment) | Organism |
---|---|
expressed as N-terminal 6× His-tagged fusion proteins under control of the T5 promoter in Escherichia coli M15(pREP4), homologous expression of wild-type and mutant M68V in Streptomyces albulus CR1 | Streptomyces albulus |
The BamHI-PstI 6.5-kb fragment is cloned and sequenced. The ApaI 1.9-kb fragment carrying the Ask genes (wild type and M68V mutant) is cloned into the pLAE003 plasmid. The resulting plasmids are used for homologous expression in Streptomyces albulus CR1. The PCR amplified fragments carrying the Ask genes (wild type and M68V mutant) with their own ribosome binding site (RBS) are cloned into the pLAE006 plasmid. The resulting plasmids are used for the homologous expression in Streptomyces albulus CR1. The pQE30 plasmid and Escherichia coli M15 (pREP4) are used to overexpress the recombinant protein. | Streptomyces albulus |
Protein Variants | Comment | Organism |
---|---|---|
I119V/M68V/T309 | random mutagenesis by error-prone PCR and subsequent site-directed mutagenesis, the mutations remove the regulation from the Ask wild type enzyme and conferre a feedback-inhibition resistance | Streptomyces albulus |
I19V/M68V/T309A | removes regulation from the Ask wild type enzyme and conferrs a feedback-inhibition resistance, assay mixture with 100 mM L-lysine plus 100 mM L-threonine reveals more than 80% activity | Streptomyces albulus |
M68V | is almost fully resistant to feedback inhibition, increase in the catalytic activity, homologous expression reveals increase in varepsilon-poly-lysine productivity | Streptomyces albulus |
M68V | random mutagenesis by error-prone PCR and subsequent site-directed mutagenesis, fully resistant to feedback inhibition, increase in epsilon-poly-L-lysine productivity | Streptomyces albulus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
3-hydroxy-L-norvaline | works as L-threonine analog | Streptomyces albulus | |
DL-3-hydroxy-L-norvaline | L-lysine-analog | Streptomyces albulus | |
L-lysine | feedback resistance of Ask, more than 20% relative activity of Ask in the assay mixture even with extremely high concentrations of 100 mM L-lysine and L-threonine each, L-Lysine alone has no effect on activity; more than 20% relative activity of rAsk is detected in the assay mixture even with extremely high concentrations of L-lysine and L-threonine (100 mM each), L-lysine alone has no effect on activity | Streptomyces albulus | |
L-threonine | feedback resistance of Ask, more than 20% relative activity of Ask in the assay mixture even with extremely high concentrations of 100 mM L-lysine and L-threonine each, L-threonine alone has no effect on activity; more than 20% activity in the rAsk is detected even with extremely high concentrations of L-lysine and L-threonine (100 mM each), L-threonine alone has no effect on activity | Streptomyces albulus | |
additional information | Ask of Streptomyces albulus CR1 is found to be the feedback resistant enzyme, but Ask of Streptomyces albulus CR1 is also subject to feedback inhibition by a mixture of L-lysine plus L-threonine of more than 1 mM each. L-lysine or L-threonine alone has no effect on activity. | Streptomyces albulus | |
S-(2-aminoethyl)-L-cysteine | L-threonine-analog; works as L-lysine analog | Streptomyces albulus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.8 | - |
L-aspartate | mutant M68V | Streptomyces albulus | |
1.8 | - |
L-aspartic acid | mutant M68V, recombinant enzyme purified to homogeneity | Streptomyces albulus | |
3 | - |
ATP | wild-type | Streptomyces albulus | |
3 | - |
ATP | wild-type, recombinant enzyme purified to homogeneity | Streptomyces albulus | |
3.7 | - |
ATP | mutant M68V | Streptomyces albulus | |
3.7 | - |
ATP | mutant M68V, recombinant enzyme purified to homogeneity | Streptomyces albulus | |
50.1 | - |
L-aspartate | wild-type | Streptomyces albulus | |
50.1 | - |
L-aspartic acid | wild-type, recombinant enzyme purified to homogeneity | Streptomyces albulus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-aspartate | Streptomyces albulus | - |
ADP + 4-phospho-L-aspartate | - |
? | |
ATP + L-aspartate | Streptomyces albulus CR1 | - |
ADP + 4-phospho-L-aspartate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces albulus | - |
- |
- |
Streptomyces albulus | Q0KJ33 | homologous expression in Streptomyces albulus CR1, expression with pQE30 plasmid in Escherichia coli M15 (pREP4) | - |
Streptomyces albulus CR1 | - |
- |
- |
Streptomyces albulus CR1 | Q0KJ33 | homologous expression in Streptomyces albulus CR1, expression with pQE30 plasmid in Escherichia coli M15 (pREP4) | - |
Purification (Comment) | Organism |
---|---|
nickel-nitriloacetic acid (Ni-NTA) affinity chromatography | Streptomyces albulus |
to near homogeneity | Streptomyces albulus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.13 | - |
wild-type | Streptomyces albulus |
5.4 | - |
mutant I19V/M68V/T309A | Streptomyces albulus |
5.4 | - |
in the absence of a mixture of L-lysine and L-threonine, mutant I19V/M68V/T309A | Streptomyces albulus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-aspartate | - |
Streptomyces albulus | ADP + 4-phospho-L-aspartate | - |
? | |
ATP + L-aspartate | - |
Streptomyces albulus CR1 | ADP + 4-phospho-L-aspartate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Ask | - |
Streptomyces albulus |
aspartokinase | - |
Streptomyces albulus |