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Literature summary for 2.7.2.4 extracted from

  • Hamano, Y.; Nicchu, I.; Shimizu, T.; Onji, Y.; Hiraki, J.; Takagi, H.
    varepsilon-Poly-L-lysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase (2007), Appl. Microbiol. Biotechnol., 76, 873-882.
    View publication on PubMed

Cloned(Commentary)

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

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

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 [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/ Products (Substrates)

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

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 (Commentary)

Purification (Comment) Organism
nickel-nitriloacetic acid (Ni-NTA) affinity chromatography Streptomyces albulus
to near homogeneity Streptomyces albulus

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
Ask
-
Streptomyces albulus
aspartokinase
-
Streptomyces albulus