BRENDA - Enzyme Database show

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

James, C.L.; Viola, R.E.; Biochemistry 41, 3720-3725 (2002)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.3
both catalytic domains of the enzyme, performing each one of the enzyme activities, are expressed separately with or without the interface region, resulting in increased activity of each domain compared to the wild-type bifunctional holoenzyme, the isolated catalytic domains are no longer allosterically regulated, expression of hybrid holoenzyme AKIII-HDHI+
Escherichia coli
2.7.2.4
both catalytic domains of the enzyme, performing each one of the enzyme activities, are expressed separately with or without the interface region, resulting in increased activity of each domain compared to the wild-type bifunctional holoenzyme, the isolated catalytic domains are no longer allosterically regulated, expression of hybrid holoenzyme AKIII-HDHI+
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.3
additional information
construction of a hybrid enzyme AKIII-HDHI+ by fusing a wild-type monofunctional aspartate kinase AKIII enzyme to the thrA2+ gene, encoding the homoserine dehydrogenase including the interface region of the wild-type bifunctional enzyme, the hybrid enzyme shows highly improved kinetic properties for homoserine dehydrogenase activity, and is not sensitive to L-threonine inhibition
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.7.2.4
L-threonine
feedback inhibition
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.3
0.41
-
L-homoserine
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
0.68
-
L-homoserine
recombinant isolated catalytic HDH-domain containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
1.2
-
L-homoserine
recombinant wild-type bifunctional holoenzyme, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
17.2
-
L-homoserine
recombinant isolated catalytic HDH-domain not containing the interface region, homoserine dehydrogenase activity
Escherichia coli
2.7.2.4
0.51
-
L-aspartate
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, asparate kinase activity
Escherichia coli
2.7.2.4
0.63
-
L-aspartate
recombinant wild-type bifunctional holoenzyme, asparate kinase activity
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
Escherichia coli
part of the aspartate pathway of amino acid biosynthesis
L-homoserine + NADP+
-
-
r
2.7.2.4
ATP + L-aspartate
Escherichia coli
part of the aspartate pathway of amino acid biosynthesis
ADP + 4-phospho-L-aspartate
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.3
Escherichia coli
-
-
-
2.7.2.4
Escherichia coli
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.3
recombinant separated catalytic domains
Escherichia coli
Reaction
EC Number
Reaction
Commentary
Organism
1.1.1.3
L-homoserine + NAD(P)+ = L-aspartate 4-semialdehyde + NAD(P)H + H+
bifunctional enzyme showing aspartate kinase, EC 2.7.2.4, and homoserine dehydrogenase activities
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
-
654640
Escherichia coli
L-homoserine + NADP+
-
-
-
r
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
part of the aspartate pathway of amino acid biosynthesis
654640
Escherichia coli
L-homoserine + NADP+
-
-
-
r
2.7.2.4
ATP + L-aspartate
part of the aspartate pathway of amino acid biosynthesis
654640
Escherichia coli
ADP + 4-phospho-L-aspartate
-
-
-
?
2.7.2.4
ATP + L-aspartate
aspartate kinase activity
654640
Escherichia coli
ADP + 4-phospho-L-aspartate
-
-
-
?
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.3
0.24
-
L-homoserine
recombinant wild-type bifunctional holoenzyme, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
0.51
-
L-homoserine
recombinant isolated catalytic HDH-domain not containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
3.3
-
L-homoserine
recombinant isolated catalytic HDH-domain containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
24
-
L-homoserine
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, homoserine dehydrogenase activity
Escherichia coli
2.7.2.4
0.16
-
L-aspartate
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, asparate kinase activity
Escherichia coli
2.7.2.4
0.39
-
L-aspartate
recombinant wild-type bifunctional holoenzyme, asparate kinase activity
Escherichia coli
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.7.2.4
0.33
-
L-threonine
recombinant wild-type bifunctional holoenzyme, inhibition of asparate kinase activity
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.3
both catalytic domains of the enzyme, performing each one of the enzyme activities, are expressed separately with or without the interface region, resulting in increased activity of each domain compared to the wild-type bifunctional holoenzyme, the isolated catalytic domains are no longer allosterically regulated, expression of hybrid holoenzyme AKIII-HDHI+
Escherichia coli
2.7.2.4
both catalytic domains of the enzyme, performing each one of the enzyme activities, are expressed separately with or without the interface region, resulting in increased activity of each domain compared to the wild-type bifunctional holoenzyme, the isolated catalytic domains are no longer allosterically regulated, expression of hybrid holoenzyme AKIII-HDHI+
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.3
additional information
construction of a hybrid enzyme AKIII-HDHI+ by fusing a wild-type monofunctional aspartate kinase AKIII enzyme to the thrA2+ gene, encoding the homoserine dehydrogenase including the interface region of the wild-type bifunctional enzyme, the hybrid enzyme shows highly improved kinetic properties for homoserine dehydrogenase activity, and is not sensitive to L-threonine inhibition
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.7.2.4
L-threonine
feedback inhibition
Escherichia coli
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.7.2.4
0.33
-
L-threonine
recombinant wild-type bifunctional holoenzyme, inhibition of asparate kinase activity
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.3
0.41
-
L-homoserine
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
0.68
-
L-homoserine
recombinant isolated catalytic HDH-domain containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
1.2
-
L-homoserine
recombinant wild-type bifunctional holoenzyme, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
17.2
-
L-homoserine
recombinant isolated catalytic HDH-domain not containing the interface region, homoserine dehydrogenase activity
Escherichia coli
2.7.2.4
0.51
-
L-aspartate
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, asparate kinase activity
Escherichia coli
2.7.2.4
0.63
-
L-aspartate
recombinant wild-type bifunctional holoenzyme, asparate kinase activity
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
Escherichia coli
part of the aspartate pathway of amino acid biosynthesis
L-homoserine + NADP+
-
-
r
2.7.2.4
ATP + L-aspartate
Escherichia coli
part of the aspartate pathway of amino acid biosynthesis
ADP + 4-phospho-L-aspartate
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.3
recombinant separated catalytic domains
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
-
654640
Escherichia coli
L-homoserine + NADP+
-
-
-
r
1.1.1.3
L-aspartate 4-semialdehyde + NADPH
part of the aspartate pathway of amino acid biosynthesis
654640
Escherichia coli
L-homoserine + NADP+
-
-
-
r
2.7.2.4
ATP + L-aspartate
part of the aspartate pathway of amino acid biosynthesis
654640
Escherichia coli
ADP + 4-phospho-L-aspartate
-
-
-
?
2.7.2.4
ATP + L-aspartate
aspartate kinase activity
654640
Escherichia coli
ADP + 4-phospho-L-aspartate
-
-
-
?
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.3
0.24
-
L-homoserine
recombinant wild-type bifunctional holoenzyme, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
0.51
-
L-homoserine
recombinant isolated catalytic HDH-domain not containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
3.3
-
L-homoserine
recombinant isolated catalytic HDH-domain containing the interface region, homoserine dehydrogenase activity
Escherichia coli
1.1.1.3
24
-
L-homoserine
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, homoserine dehydrogenase activity
Escherichia coli
2.7.2.4
0.16
-
L-aspartate
recombinant hybrid bifunctional holoenzyme AKIII-HDHI+ containing the interface region, asparate kinase activity
Escherichia coli
2.7.2.4
0.39
-
L-aspartate
recombinant wild-type bifunctional holoenzyme, asparate kinase activity
Escherichia coli