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Literature summary extracted from

  • Lee, S.C.; Jung, I.P.; Baig, I.A.; Chien, P.N.; La, I.J.; Yoon, M.Y.
    Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583 (2015), Int. J. Biol. Macromol., 72, 104-109.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.2.1.6 additional information the enzyme is a FAD-independent catabolic enzyme form Enterococcus faecalis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.2.1.6 expressed in Escherichia coli BL21(DE3) cells Enterococcus faecalis
2.2.1.6 gene als, genetic location within the butanediol operon, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Enterococcus faecalis

Protein Variants

EC Number Protein Variants Comment Organism
2.2.1.6 H111F site-directed mutagenesis, the mutant enzyme exhibits about 50fold lower specific activity with significant reduction in kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 H111F the mutant displays 26fold increase in Km value compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 H111R site-directed mutagenesis, the mutant enzyme exhibits increasedspecific activity and kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 H111R the mutant displays 17fold increase in Km value compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 Q112E site-directed mutagenesis, the mutant enzyme exhibits about 50fold lower specific activity with significant reduction in kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 Q112E the mutant exhibits significantly lower specific activity with 70fold higher Ks for thiamine diphosphate compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 Q112N site-directed mutagenesis, the mutant enzyme exhibits about 50fold lower specific activity with significant reduction in kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 Q112N the mutant exhibits significantly lower specific activity with 15fold higher Ks for thiamine diphosphate compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 Q112V site-directed mutagenesis, the mutant enzyme exhibits about 50fold lower specific activity with significant reduction in kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 Q112V the mutant exhibits significantly lower specific activity with 10fold higher Ks for thiamine diphosphate compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 Q411E site-directed mutagenesis, the mutant enzyme exhibits increased specific activity and kcat compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 Q411E the mutant shows a 10fold rise in Km and a 20fold increase in Ks for thiamine diphosphate compared to the wild type enzyme Enterococcus faecalis
2.2.1.6 Q411N site-directed mutagenesis, the mutant enzyme has a 3fold increased Km compared to the wild-type enzyme Enterococcus faecalis
2.2.1.6 Q411N the mutant exhibits increased specific activity and kcat compared to the wild type enzyme Enterococcus faecalis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.2.1.6 additional information
-
 additional information binding kinetics of Mg2+ and thiamine diphosphate with wild-type enzyme and mutant enzymes, overview Enterococcus faecalis
2.2.1.6 1.37
-
 pyruvate pH 7.5, 37°C, recombinant wild-type enzyme Enterococcus faecalis
2.2.1.6 1.37
-
 pyruvate wild type enzyme, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 4.34
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q411N Enterococcus faecalis
2.2.1.6 4.34
-
 pyruvate mutant enzyme Q411N, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 14.68
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112N Enterococcus faecalis
2.2.1.6 14.68
-
 pyruvate mutant enzyme Q112N, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 16.43
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q411E Enterococcus faecalis
2.2.1.6 16.43
-
 pyruvate mutant enzyme Q411E, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 19.78
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112E Enterococcus faecalis
2.2.1.6 19.78
-
 pyruvate mutant enzyme Q112E, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 24.6
-
 pyruvate pH 7.5, 37°C, recombinant mutant H111R Enterococcus faecalis
2.2.1.6 24.6
-
 pyruvate mutant enzyme H111R, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 31.2
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112V Enterococcus faecalis
2.2.1.6 31.2
-
 pyruvate mutant enzyme Q112V, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 36.6
-
 pyruvate pH 7.5, 37°C, recombinant mutant H111F Enterococcus faecalis
2.2.1.6 36.6
-
 pyruvate mutant enzyme H111F, at pH 6.8 and 37°C Enterococcus faecalis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.2.1.6 Mg2+ divalent metal ion Mg2+ i required for catalytic activity. In catalysis, a divalent metal ion Mg2+ serves to anchor the diphosphate moiety of thiamine diphosphate at the active site of the catabolic enzyme Enterococcus faecalis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.2.1.6 60000
-
 x * 60000, SDS-PAGE Enterococcus faecalis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.2.1.6 2 pyruvate Enterococcus faecalis
-
 2-acetolactate + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.2.1.6 Enterococcus faecalis Q836A5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.2.1.6 Ni+-chelating column chromatography Enterococcus faecalis
2.2.1.6 recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Enterococcus faecalis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.2.1.6 2 pyruvate
-
 Enterococcus faecalis 2-acetolactate + CO2
-
 ?

Subunits

EC Number Subunits Comment Organism
2.2.1.6 ? x * 60000, SDS-PAGE Enterococcus faecalis
2.2.1.6 More the catabolic enzyme form lacks a regulatory subunit Enterococcus faecalis

Synonyms

EC Number Synonyms Comment Organism
2.2.1.6 ALS
-
 Enterococcus faecalis
2.2.1.6 CalS
-
 Enterococcus faecalis
2.2.1.6 catabolic acetolactate synthase
-
 Enterococcus faecalis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.2.1.6 37
-
 assay at Enterococcus faecalis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.2.1.6 0.08
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112E Enterococcus faecalis
2.2.1.6 0.08
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112V Enterococcus faecalis
2.2.1.6 0.08
-
 pyruvate mutant enzyme Q112E, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 0.08
-
 pyruvate mutant enzyme Q112V, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 0.22
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q112N Enterococcus faecalis
2.2.1.6 0.22
-
 pyruvate mutant enzyme Q112N, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 0.49
-
 pyruvate pH 7.5, 37°C, recombinant mutant H111F Enterococcus faecalis
2.2.1.6 0.49
-
 pyruvate mutant enzyme H111F, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 2.88
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q411E Enterococcus faecalis
2.2.1.6 2.88
-
 pyruvate mutant enzyme Q411E, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 5.28
-
 pyruvate pH 7.5, 37°C, recombinant wild-type enzyme Enterococcus faecalis
2.2.1.6 5.28
-
 pyruvate wild type enzyme, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 15.9
-
 pyruvate pH 7.5, 37°C, recombinant mutant Q411N Enterococcus faecalis
2.2.1.6 15.9
-
 pyruvate mutant enzyme Q411N, at pH 6.8 and 37°C Enterococcus faecalis
2.2.1.6 28.6
-
 pyruvate pH 7.5, 37°C, recombinant mutant H111R Enterococcus faecalis
2.2.1.6 28.6
-
 pyruvate mutant enzyme H111R, at pH 6.8 and 37°C Enterococcus faecalis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.2.1.6 6
-
-
 Enterococcus faecalis

Cofactor

EC Number Cofactor Comment Organism Structure
2.2.1.6 additional information FAD-independent enzyme Enterococcus faecalis
2.2.1.6 thiamine diphosphate
-
 Enterococcus faecalis
2.2.1.6 thiamine diphosphate dependent on, located centrally in the active site of cALS with a unique V-conformation at the dimer interface to play a central role of intramolecular protontransfer in the catalytic cycle. In catalysis, a divalent metal ion Mg2+ serves to anchor the diphosphate moiety of thiamine diphosphate at the active site of the catalbolic enzyme Enterococcus faecalis

General Information

EC Number General Information Comment Organism
2.2.1.6 additional information the catabolic enzyme form lacks a regulatory subunit. Residue His 111, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding, importance of residues H111, Q112, and Q411 residues for catalysis, Q112 and Q411 might be involved in thiamine diphosphate binding, enzyme structure homology modeling, overview Enterococcus faecalis
2.2.1.6 physiological function catabolic acetolactate synthase from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate Enterococcus faecalis