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Literature summary for 2.2.1.6 extracted from

  • Baig, I.A.; Moon, J.Y.; Kim, M.S.; Koo, B.S.; Yoon, M.Y.
    Structural and functional significance of the highly-conserved residues in Mycobacterium tuberculosis acetohydroxyacid synthase (2014), Enzyme Microb. Technol., 58-59, 52-59.
    View publication on PubMed
Search for more literature for 2.2.1.6

Application

Application Comment Organism
drug development the enzyme is a target for the development of anti-tuberculosis agents Mycobacterium tuberculosis

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Mycobacterium tuberculosis
recombinant expression of the wild-type enzyme's catalytic subunit, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Protein Variants

Protein Variants Comment Organism
E85A site-directed mutagenesis, the mutation leads to severe drop in catalyticactivity with reduced affinity toward thiamine diphosphate, , the enzyme shows reduced activity compared to the wild-type enzyme Mycobacterium tuberculosis
E85A the mutation leads to severe drop in catalytic activity (0.08% of wild type activity) with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
E85A the mutation leads to severe drop in catalytic activity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
E85D site-directed mutagenesis, the mutation leads to severe drop in catalyticactivity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
E85D the mutation leads to severe drop in catalytic activity (2.23% of wild type activity) with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
E85Q site-directed mutagenesis, the mutation leads to severe drop in catalyticactivity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
E85Q the mutation leads to severe drop in catalytic activity (1.2% of wild type activity) with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
H84A site-directed mutagenesis, the mutation leads to the loss of many hydrogen bonds among residues His84, Glu85, and Gln86 in wild-type enzyme Mycobacterium tuberculosis
H84A the mutation leads to severe drop in catalytic activity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
H84T site-directed mutagenesis, the enzyme shows reduced activity compared to the wild-type enzyme Mycobacterium tuberculosis
H84T the mutation leads to severe drop in catalytic activity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
Q86A site-directed mutagenesis, the enzyme shows reduced activity compared to the wild-type enzyme Mycobacterium tuberculosis
Q86A the mutation leads to severe drop in catalytic activity with reduced affinity toward thiamine diphosphate Mycobacterium tuberculosis
Q86W site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis
Q86W the mutation completely abolishes the enzyme's activity Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0028
-
 pyruvate wild type enzyme, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.0028
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant H84A Mycobacterium tuberculosis
0.0028
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant H84T Mycobacterium tuberculosis
0.0028
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant Q86A Mycobacterium tuberculosis
0.0028
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged wild-type enzyme Mycobacterium tuberculosis
0.141
-
 pyruvate mutant enzyme Q86A, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.15
-
 pyruvate mutant enzyme H84A, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.213
-
 pyruvate mutant enzyme H84T, at pH 7.5 and 37°C Mycobacterium tuberculosis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ the enzyme requires a divalent metal ion such as Mg2+ to anchor thiamine diphosphate Mycobacterium tuberculosis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
68000
-
 x * 68000, SDS-PAGE Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2 pyruvate Mycobacterium tuberculosis
-
 2-acetolactate + CO2
-
 ?
2 pyruvate Mycobacterium tuberculosis
-
 2-acetolactate + CO2
-
 ir
2 pyruvate Mycobacterium tuberculosis H37Rv
-
 2-acetolactate + CO2
-
 ?
2 pyruvate Mycobacterium tuberculosis H37Rv
-
 2-acetolactate + CO2
-
 ir
pyruvate + 2-oxobutyrate Mycobacterium tuberculosis
-
 2-aceto-2-hydroxybutyrate + CO2
-
 ir
pyruvate + 2-oxobutyrate Mycobacterium tuberculosis H37Rv
-
 2-aceto-2-hydroxybutyrate + CO2
-
 ir

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis P9WG41
-
-
Mycobacterium tuberculosis H37Rv P9WG41
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Mycobacterium tuberculosis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0023
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant E85A Mycobacterium tuberculosis
0.034
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant E85Q Mycobacterium tuberculosis
0.04
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant H84A Mycobacterium tuberculosis
0.063
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant E85D Mycobacterium tuberculosis
0.19
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant H84T Mycobacterium tuberculosis
0.22
-
 pH 7.5, 37°C, purified recombinant His6-tagged mutant Q86A Mycobacterium tuberculosis
2.8
-
 pH 7.5, 37°C, purified recombinant His6-tagged wild-type enzyme Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 pyruvate
-
 Mycobacterium tuberculosis 2-acetolactate + CO2
-
 ?
2 pyruvate
-
 Mycobacterium tuberculosis 2-acetolactate + CO2
-
 ir
2 pyruvate
-
 Mycobacterium tuberculosis H37Rv 2-acetolactate + CO2
-
 ?
2 pyruvate
-
 Mycobacterium tuberculosis H37Rv 2-acetolactate + CO2
-
 ir
pyruvate + 2-oxobutyrate
-
 Mycobacterium tuberculosis 2-aceto-2-hydroxybutyrate + CO2
-
 ir
pyruvate + 2-oxobutyrate
-
 Mycobacterium tuberculosis H37Rv 2-aceto-2-hydroxybutyrate + CO2
-
 ir

Subunits

Subunits Comment Organism
? x * 68000, SDS-PAGE Mycobacterium tuberculosis
More the enzyme is comprised of two subunits: a large catalytic subunit and asmall regulatory one Mycobacterium tuberculosis

Synonyms

Synonyms Comment Organism
acetohydroxyacid synthase
-
 Mycobacterium tuberculosis
AHAS
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0026
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant E85A Mycobacterium tuberculosis
0.0026
-
 pyruvate mutant enzyme E85A, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.038
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant E85Q Mycobacterium tuberculosis
0.0384
-
 pyruvate mutant enzyme E85Q, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.046
-
 pyruvate mutant enzyme H84A, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.05
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant H84A Mycobacterium tuberculosis
0.071
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant E85D Mycobacterium tuberculosis
0.0712
-
 pyruvate mutant enzyme E85D, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.21
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant H84T Mycobacterium tuberculosis
0.212
-
 pyruvate mutant enzyme H84T, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.25
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged mutant Q86A Mycobacterium tuberculosis
0.25
-
 pyruvate mutant enzyme Q86A, at pH 7.5 and 37°C Mycobacterium tuberculosis
3.196
-
 pyruvate wild type enzyme, at pH 7.5 and 37°C Mycobacterium tuberculosis
3.2
-
 pyruvate pH 7.5, 37°C, recombinant His6-tagged wild-type enzyme Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Mycobacterium tuberculosis

Cofactor

Cofactor Comment Organism Structure
FAD dependent on Mycobacterium tuberculosis
thiamine diphosphate
-
 Mycobacterium tuberculosis
thiamine diphosphate dependent on Mycobacterium tuberculosis

General Information

General Information Comment Organism
malfunction the conserved His84 and Gln86 residues lie in the catalytic dimer interface of the enzyme. Mutational analyses of these invariants lead to significant reduction in their activity with reduced affinity toward the substrate Mycobacterium tuberculosis
metabolism acetohydroxyacid synthase or asacetolactate synthase catalyzes the first step in the biosynthe-sis of branched-chain amino acids such as isoleucine, leucine, and valine. This reaction involves synthesizing either (2S)-acetolactate from two molecules of pyruvate or (2S)-2-aceto-2-hydroxybutyrate from pyruvate and 2-oxobutyrate Mycobacterium tuberculosis
additional information homology modeling of Mycobacterium tuberculosis enzyme is performed by using crystal structures, PDB IDs 1N0H and 1JSC, from the Saccharomyces cerevisiae enzyme as template, molcular dynamics simulation Mycobacterium tuberculosis
additional information molecular dynamics simulation studies suggest that the conserved His84 and Gln86 residues residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1'atom of thiamine diphosphate during catalysis Mycobacterium tuberculosis