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Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32

Zhang, W.W.; Jiang, W.H.; Zhao, G.P.; Yang, Y.L.; Chiao, J.S.; Appl. Microbiol. Biotechnol. 54, 52-58 (2000)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.2.1.11
expressed in Escherichia coli
Amycolatopsis mediterranei U32
2.7.2.4
operon encoding aspartokinase cloned and sequenced, introduced into auxotrophic Escherichia coli CGSC5074 and Escherichia coli X6118 and functionally expressed
Amycolatopsis mediterranei
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.2.1.11
L-isoleucine
at 1 mM: 18% repression of enzyme synthesis, at 5 mM: 62% repression of enzyme synthesis
Amycolatopsis mediterranei U32
1.2.1.11
L-lysine
at 1 mM: 22% repression of the enzyme synthesis, at 5 mM: 44% repression of the enzyme synthesis, at 10 mM: 28% inhibition of the enzyme activity assayed in the reverse reaction
Amycolatopsis mediterranei U32
1.2.1.11
L-methionine
at 1 mM: 40% repression of the enzyme synthesis, at 5 mM: 62% repression of the enzyme synthesis
Amycolatopsis mediterranei U32
1.2.1.11
L-threonine
at 1 mM: 25% repression of the enzyme synthesis, at 5 mM: 43% repression of the enzyme synthesis, at 10 mM: 37% inhibition of the enzyme activity assayed in the reverse reaction
Amycolatopsis mediterranei U32
2.7.2.4
L-isoleucine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
2.7.2.4
L-lysine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
2.7.2.4
L-threonine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.73
-
NADP+
-
Amycolatopsis mediterranei U32
1.2.1.11
1.25
-
L-aspartate 4-semialdehyde
-
Amycolatopsis mediterranei U32
2.7.2.4
2.3
-
ATP
-
Amycolatopsis mediterranei
2.7.2.4
3.4
-
L-aspartate
-
Amycolatopsis mediterranei
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.2.1.11
37000
-
x * 37000, SDS-PAGE
Amycolatopsis mediterranei U32
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.2.1.11
Amycolatopsis mediterranei U32
-
-
-
2.7.2.4
Amycolatopsis mediterranei
-
U32
-
Purification (Commentary)
EC Number
Commentary
Organism
1.2.1.11
-
Amycolatopsis mediterranei U32
2.7.2.4
recombinant enzyme, expressed in E. coli
Amycolatopsis mediterranei
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
1.2.1.11
0.0029
-
of the recombinant in the crude extracts of Escherichia coli
Amycolatopsis mediterranei U32
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 + phosphate + NADP+
-
390201
Amycolatopsis mediterranei U32
L-4-aspartyl phosphate + NADPH
-
390201
Amycolatopsis mediterranei U32
-
Subunits
EC Number
Subunits
Commentary
Organism
1.2.1.11
?
x * 37000, SDS-PAGE
Amycolatopsis mediterranei U32
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Amycolatopsis mediterranei U32
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
expressed in Escherichia coli
Amycolatopsis mediterranei U32
2.7.2.4
operon encoding aspartokinase cloned and sequenced, introduced into auxotrophic Escherichia coli CGSC5074 and Escherichia coli X6118 and functionally expressed
Amycolatopsis mediterranei
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Amycolatopsis mediterranei U32
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.2.1.11
L-isoleucine
at 1 mM: 18% repression of enzyme synthesis, at 5 mM: 62% repression of enzyme synthesis
Amycolatopsis mediterranei U32
1.2.1.11
L-lysine
at 1 mM: 22% repression of the enzyme synthesis, at 5 mM: 44% repression of the enzyme synthesis, at 10 mM: 28% inhibition of the enzyme activity assayed in the reverse reaction
Amycolatopsis mediterranei U32
1.2.1.11
L-methionine
at 1 mM: 40% repression of the enzyme synthesis, at 5 mM: 62% repression of the enzyme synthesis
Amycolatopsis mediterranei U32
1.2.1.11
L-threonine
at 1 mM: 25% repression of the enzyme synthesis, at 5 mM: 43% repression of the enzyme synthesis, at 10 mM: 37% inhibition of the enzyme activity assayed in the reverse reaction
Amycolatopsis mediterranei U32
2.7.2.4
L-isoleucine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
2.7.2.4
L-lysine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
2.7.2.4
L-threonine
repression of aspartokinase gene transcription
Amycolatopsis mediterranei
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2.1.11
0.73
-
NADP+
-
Amycolatopsis mediterranei U32
1.2.1.11
1.25
-
L-aspartate 4-semialdehyde
-
Amycolatopsis mediterranei U32
2.7.2.4
2.3
-
ATP
-
Amycolatopsis mediterranei
2.7.2.4
3.4
-
L-aspartate
-
Amycolatopsis mediterranei
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.2.1.11
37000
-
x * 37000, SDS-PAGE
Amycolatopsis mediterranei U32
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
-
Amycolatopsis mediterranei U32
2.7.2.4
recombinant enzyme, expressed in E. coli
Amycolatopsis mediterranei
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
1.2.1.11
0.0029
-
of the recombinant in the crude extracts of Escherichia coli
Amycolatopsis mediterranei U32
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 + phosphate + NADP+
-
390201
Amycolatopsis mediterranei U32
L-4-aspartyl phosphate + NADPH
-
390201
Amycolatopsis mediterranei U32
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.2.1.11
?
x * 37000, SDS-PAGE
Amycolatopsis mediterranei U32