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Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis

Vyas, R.; Kumar, V.; Panjikar, S.; Karthikeyan, S.; Kishan, K.V.; Tewari, R.; Weiss, M.S.; Acta Crystallogr. Sect. F 64, 167-170 (2008)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.2.1.11
expression of His-tagged enzyme in Escherichia coli strain M15; heterologous expression in Escherichia coli
Mycobacterium tuberculosis
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.2.1.11
by using the hanging-drop vapour-diffusion method, crystallization in 2 different crystal forms, diffraction data analysis suggests the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form and of one or 2 monomers in the cubic crystal form; purified recombinant His-tagged enzyme, 9 mg/ml protein in 10 mM potassium phosphate buffer, pH 8.0, and 10 mM DTT, sitting drop vapour diffusion method, mixing of 500 nl of protein and of reservoir solution, the latter containing 1.6 M ammonium sulfate and 100 mM citric acid, pH 5.0, 2 days at 18°C, two different crystal forms, X-ray diffraction structure determination and analysis at 2.18-2.75 A resolution
Mycobacterium tuberculosis
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.2.1.11
37000
-
2 * 37000, about, recombinant His-tagged nezyme, SDS-PAGE
Mycobacterium tuberculosis
1.2.1.11
38070
-
the expressed and purified monomer protein construct includes the 17-amino-acid sequence RGSHHHHHHGSACELGT between the N-terminal Met and the second amino acid Gly of the native Asd sequence, the total length of this protein is 362 amino acids
Mycobacterium tuberculosis
1.2.1.11
75000
-
homodimer in solution, determined by gel filtration; recombinant His-tagged enzyme, gel filtration
Mycobacterium tuberculosis
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis
second enzyme in the lysine/homoserine biosynthetic pathways
L-4-aspartyl phosphate + NADPH + H+
-
-
r
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis H37Rv
second enzyme in the lysine/homoserine biosynthetic pathways
L-4-aspartyl phosphate + NADPH + H+
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis
the enzyme takes part in the lysine/homoserine-biosynthetic pathway
L-4-aspartyl phosphate + NADPH + H+
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.2.1.11
Mycobacterium tuberculosis
-
gene Rv3708c
-
1.2.1.11
Mycobacterium tuberculosis
P9WNX5
-
-
1.2.1.11
Mycobacterium tuberculosis H37Rv
P9WNX5
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.2.1.11
by using affinity chromatography and gel filtration; recombinant His-tagged enzyme from Escherichia coli strain M15 by nickel affinity chromatography and gel filtration
Mycobacterium tuberculosis
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
second enzyme in the lysine/homoserine biosynthetic pathways
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
second enzyme in the lysine/homoserine biosynthetic pathways
684192
Mycobacterium tuberculosis H37Rv
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
-
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
?
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
the enzyme takes part in the lysine/homoserine-biosynthetic pathway
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.2.1.11
dimer
2 * 37000, about, recombinant His-tagged nezyme, SDS-PAGE
Mycobacterium tuberculosis
1.2.1.11
homodimer
recombinant enzyme
Mycobacterium tuberculosis
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Mycobacterium tuberculosis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
expression of His-tagged enzyme in Escherichia coli strain M15
Mycobacterium tuberculosis
1.2.1.11
heterologous expression in Escherichia coli
Mycobacterium tuberculosis
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.2.1.11
NADP+
-
Mycobacterium tuberculosis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.2.1.11
by using the hanging-drop vapour-diffusion method, crystallization in 2 different crystal forms, diffraction data analysis suggests the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form and of one or 2 monomers in the cubic crystal form
Mycobacterium tuberculosis
1.2.1.11
purified recombinant His-tagged enzyme, 9 mg/ml protein in 10 mM potassium phosphate buffer, pH 8.0, and 10 mM DTT, sitting drop vapour diffusion method, mixing of 500 nl of protein and of reservoir solution, the latter containing 1.6 M ammonium sulfate and 100 mM citric acid, pH 5.0, 2 days at 18°C, two different crystal forms, X-ray diffraction structure determination and analysis at 2.18-2.75 A resolution
Mycobacterium tuberculosis
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.2.1.11
37000
-
2 * 37000, about, recombinant His-tagged nezyme, SDS-PAGE
Mycobacterium tuberculosis
1.2.1.11
38070
-
the expressed and purified monomer protein construct includes the 17-amino-acid sequence RGSHHHHHHGSACELGT between the N-terminal Met and the second amino acid Gly of the native Asd sequence, the total length of this protein is 362 amino acids
Mycobacterium tuberculosis
1.2.1.11
75000
-
homodimer in solution, determined by gel filtration
Mycobacterium tuberculosis
1.2.1.11
75000
-
recombinant His-tagged enzyme, gel filtration
Mycobacterium tuberculosis
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis
second enzyme in the lysine/homoserine biosynthetic pathways
L-4-aspartyl phosphate + NADPH + H+
-
-
r
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis H37Rv
second enzyme in the lysine/homoserine biosynthetic pathways
L-4-aspartyl phosphate + NADPH + H+
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
Mycobacterium tuberculosis
the enzyme takes part in the lysine/homoserine-biosynthetic pathway
L-4-aspartyl phosphate + NADPH + H+
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.2.1.11
by using affinity chromatography and gel filtration
Mycobacterium tuberculosis
1.2.1.11
recombinant His-tagged enzyme from Escherichia coli strain M15 by nickel affinity chromatography and gel filtration
Mycobacterium tuberculosis
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
aspartate-4-semialdehyde + phosphate + NADP+
second enzyme in the lysine/homoserine biosynthetic pathways
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
1.2.1.11
aspartate-4-semialdehyde + phosphate + NADP+
second enzyme in the lysine/homoserine biosynthetic pathways
684192
Mycobacterium tuberculosis H37Rv
L-4-aspartyl phosphate + NADPH + H+
-
-
-
r
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
-
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
?
1.2.1.11
L-aspartate 4-semialdehyde + phosphate + NADP+
the enzyme takes part in the lysine/homoserine-biosynthetic pathway
684192
Mycobacterium tuberculosis
L-4-aspartyl phosphate + NADPH + H+
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.2.1.11
dimer
2 * 37000, about, recombinant His-tagged nezyme, SDS-PAGE
Mycobacterium tuberculosis
1.2.1.11
homodimer
recombinant enzyme
Mycobacterium tuberculosis