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Literature summary for 2.2.1.6 extracted from

  • Baig, I.A.; Gedi, V.; Lee, S.C.; Koh, S.H.; Yoon, M.Y.
    Role of a highly conserved proline-126 in ThDP binding of Mycobacterium tuberculosis acetohydroxyacid synthase (2013), Enzyme Microb. Technol., 53, 243-249.
    View publication on PubMed

Application

Application Comment Organism
drug development acetohydroxyacid synthase (AHAS) of Mycobacterium tuberculosis is a promising target for the development of anti-tuberculosis agents Mycobacterium tuberculosis

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Mycobacterium tuberculosis
sequence comparisoons, recombinant expression of soluble His-tagged enzyme mutants in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Protein Variants

Protein Variants Comment Organism
P126A site-directed mutagenesis, the mutant exhibits similar kinetics but significantly lower activity compared to the wild-type enzyme Mycobacterium tuberculosis
P126A, the mutant exhibits significantly lower activity than the wild type enzyme Mycobacterium tuberculosis
P126E inactive Mycobacterium tuberculosis
P126E site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis
P126T site-directed mutagenesis, the mutant exhibits significantly lower activity than wild-type enzyme and a significantly decreased preference toward thiamine diphosphate as cofactor Mycobacterium tuberculosis
P126T the mutant exhibits significantly lower activity than the wild type enzyme Mycobacterium tuberculosis
P126V site-directed mutagenesis, the mutant exhibits significantly lower activity than wild-type enzyme and a significantly decreased preference toward pyruvate as substrate Mycobacterium tuberculosis
P126V the mutant exhibits significantly lower activity than the wild type enzyme Mycobacterium tuberculosis

Inhibitors

Inhibitors Comment Organism Structure
chlorimuron ethyl a sulfonylurea derivative herbicide Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.76
-
pyruvate pH 7.5, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis
2.76
-
pyruvate wild type enzyme, at pH 7.5 and 37°C Mycobacterium tuberculosis
6.04
-
pyruvate pH 7.5, 37°c, recombinant mutant P126A Mycobacterium tuberculosis
6.04
-
pyruvate mutant enzyme P126A, at pH 7.5 and 37°C Mycobacterium tuberculosis
466.7
-
pyruvate pH 7.5, 37°c, recombinant mutant P126V Mycobacterium tuberculosis
466.7
-
pyruvate mutant enzyme P126V, at pH 7.5 and 37°C Mycobacterium tuberculosis
807.7
-
pyruvate pH 7.5, 37°c, recombinant mutant P126T Mycobacterium tuberculosis
807.7
-
pyruvate mutant enzyme P126T, at pH 7.5 and 37°C Mycobacterium tuberculosis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Mycobacterium tuberculosis
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
68000
-
x * 68000, about, wild-type and mutant enzyme variants, 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
-
ir
2 pyruvate Mycobacterium tuberculosis
-
2-acetolactate + CO2
-
?
2 pyruvate Mycobacterium tuberculosis H37Rv
-
2-acetolactate + CO2
-
ir
2 pyruvate Mycobacterium tuberculosis H37Rv
-
2-acetolactate + CO2
-
?
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
-
-
-
Mycobacterium tuberculosis P9WG39
-
-
Mycobacterium tuberculosis H37Rv
-
-
-
Mycobacterium tuberculosis H37Rv P9WG39
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Mycobacterium tuberculosis
recombinant soluble His-tagged enzyme mutants from Escherichia coli strain BL21(DE3) to near homogeneity by nickel affinity chromatography, gel filtration, and anion exchange chromatography Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 pyruvate
-
Mycobacterium tuberculosis 2-acetolactate + CO2
-
ir
2 pyruvate
-
Mycobacterium tuberculosis 2-acetolactate + CO2
-
?
2 pyruvate
-
Mycobacterium tuberculosis H37Rv 2-acetolactate + CO2
-
ir
2 pyruvate
-
Mycobacterium tuberculosis H37Rv 2-acetolactate + CO2
-
?
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
? x * 68000, about, wild-type and mutant enzyme variants, SDS-PAGE 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
0.164
-
pyruvate pH 7.5, 37°c, recombinant mutant P126V Mycobacterium tuberculosis
0.164
-
pyruvate mutant enzyme P126V, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.165
-
pyruvate pH 7.5, 37°c, recombinant mutant P126A Mycobacterium tuberculosis
0.165
-
pyruvate mutant enzyme P126A, at pH 7.5 and 37°C Mycobacterium tuberculosis
0.166
-
pyruvate pH 7.5, 37°c, recombinant mutant P126T Mycobacterium tuberculosis
0.166
-
pyruvate mutant enzyme P126T, at pH 7.5 and 37°C Mycobacterium tuberculosis
3.196
-
pyruvate pH 7.5, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis
3.196
-
pyruvate wild type enzyme, at pH 7.5 and 37°C 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, the cofactor plays a key role in catalysis. The thiamine diphosphate binding pocket contains the highly conserved proline 126 residue, binding pocket structure, overview. Thiamine diphosphate is located centrally in the active site of AHAS with a unique V-conformation at the dimer interface. In the dimeric structure, one subunit is in contact with the diphosphate moiety of thiamine diphosphate, and the other subunit is in contact with the aminopyrimidine moiety Mycobacterium tuberculosis

General Information

General Information Comment Organism
metabolism the enzyme is involved in the branched chain amino acid biosynthesis Mycobacterium tuberculosis
additional information structure homology modeling Mycobacterium tuberculosis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.205
-
pyruvate pH 7.5, 37°c, recombinant mutant P126T Mycobacterium tuberculosis
0.352
-
pyruvate pH 7.5, 37°c, recombinant mutant P126V Mycobacterium tuberculosis
27.22
-
pyruvate pH 7.5, 37°c, recombinant mutant P126A Mycobacterium tuberculosis
1158
-
pyruvate pH 7.5, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis