Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme is a FAD-independent catabolic enzyme form | Enterococcus faecalis |
Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Enterococcus faecalis |
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 | Comment | Organism |
---|---|---|
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 |
H111F | the mutant displays 26fold increase in Km value compared to the wild type enzyme | Enterococcus faecalis |
H111R | site-directed mutagenesis, the mutant enzyme exhibits increasedspecific activity and kcat compared to the wild-type enzyme | Enterococcus faecalis |
H111R | the mutant displays 17fold increase in Km value compared to the wild type enzyme | Enterococcus faecalis |
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 |
Q112E | the mutant exhibits significantly lower specific activity with 70fold higher Ks for thiamine diphosphate compared to the wild type enzyme | Enterococcus faecalis |
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 |
Q112N | the mutant exhibits significantly lower specific activity with 15fold higher Ks for thiamine diphosphate compared to the wild type enzyme | Enterococcus faecalis |
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 |
Q112V | the mutant exhibits significantly lower specific activity with 10fold higher Ks for thiamine diphosphate compared to the wild type enzyme | Enterococcus faecalis |
Q411E | site-directed mutagenesis, the mutant enzyme exhibits increased specific activity and kcat compared to the wild-type enzyme | Enterococcus faecalis |
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 |
Q411N | site-directed mutagenesis, the mutant enzyme has a 3fold increased Km compared to the wild-type enzyme | Enterococcus faecalis |
Q411N | the mutant exhibits increased specific activity and kcat compared to the wild type enzyme | Enterococcus faecalis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | binding kinetics of Mg2+ and thiamine diphosphate with wild-type enzyme and mutant enzymes, overview | Enterococcus faecalis | |
1.37 | - |
pyruvate | pH 7.5, 37°C, recombinant wild-type enzyme | Enterococcus faecalis | |
1.37 | - |
pyruvate | wild type enzyme, at pH 6.8 and 37°C | Enterococcus faecalis | |
4.34 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q411N | Enterococcus faecalis | |
4.34 | - |
pyruvate | mutant enzyme Q411N, at pH 6.8 and 37°C | Enterococcus faecalis | |
14.68 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112N | Enterococcus faecalis | |
14.68 | - |
pyruvate | mutant enzyme Q112N, at pH 6.8 and 37°C | Enterococcus faecalis | |
16.43 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q411E | Enterococcus faecalis | |
16.43 | - |
pyruvate | mutant enzyme Q411E, at pH 6.8 and 37°C | Enterococcus faecalis | |
19.78 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112E | Enterococcus faecalis | |
19.78 | - |
pyruvate | mutant enzyme Q112E, at pH 6.8 and 37°C | Enterococcus faecalis | |
24.6 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant H111R | Enterococcus faecalis | |
24.6 | - |
pyruvate | mutant enzyme H111R, at pH 6.8 and 37°C | Enterococcus faecalis | |
31.2 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112V | Enterococcus faecalis | |
31.2 | - |
pyruvate | mutant enzyme Q112V, at pH 6.8 and 37°C | Enterococcus faecalis | |
36.6 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant H111F | Enterococcus faecalis | |
36.6 | - |
pyruvate | mutant enzyme H111F, at pH 6.8 and 37°C | Enterococcus faecalis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
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] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
60000 | - |
x * 60000, SDS-PAGE | Enterococcus faecalis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 pyruvate | Enterococcus faecalis | - |
2-acetolactate + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Enterococcus faecalis | Q836A5 | - |
- |
Purification (Comment) | Organism |
---|---|
Ni+-chelating column chromatography | Enterococcus faecalis |
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) | Enterococcus faecalis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 pyruvate | - |
Enterococcus faecalis | 2-acetolactate + CO2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 60000, SDS-PAGE | Enterococcus faecalis |
More | the catabolic enzyme form lacks a regulatory subunit | Enterococcus faecalis |
Synonyms | Comment | Organism |
---|---|---|
ALS | - |
Enterococcus faecalis |
CalS | - |
Enterococcus faecalis |
catabolic acetolactate synthase | - |
Enterococcus faecalis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Enterococcus faecalis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.08 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112E | Enterococcus faecalis | |
0.08 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112V | Enterococcus faecalis | |
0.08 | - |
pyruvate | mutant enzyme Q112E, at pH 6.8 and 37°C | Enterococcus faecalis | |
0.08 | - |
pyruvate | mutant enzyme Q112V, at pH 6.8 and 37°C | Enterococcus faecalis | |
0.22 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q112N | Enterococcus faecalis | |
0.22 | - |
pyruvate | mutant enzyme Q112N, at pH 6.8 and 37°C | Enterococcus faecalis | |
0.49 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant H111F | Enterococcus faecalis | |
0.49 | - |
pyruvate | mutant enzyme H111F, at pH 6.8 and 37°C | Enterococcus faecalis | |
2.88 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q411E | Enterococcus faecalis | |
2.88 | - |
pyruvate | mutant enzyme Q411E, at pH 6.8 and 37°C | Enterococcus faecalis | |
5.28 | - |
pyruvate | pH 7.5, 37°C, recombinant wild-type enzyme | Enterococcus faecalis | |
5.28 | - |
pyruvate | wild type enzyme, at pH 6.8 and 37°C | Enterococcus faecalis | |
15.9 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant Q411N | Enterococcus faecalis | |
15.9 | - |
pyruvate | mutant enzyme Q411N, at pH 6.8 and 37°C | Enterococcus faecalis | |
28.6 | - |
pyruvate | pH 7.5, 37°C, recombinant mutant H111R | Enterococcus faecalis | |
28.6 | - |
pyruvate | mutant enzyme H111R, at pH 6.8 and 37°C | Enterococcus faecalis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
- |
Enterococcus faecalis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | FAD-independent enzyme | Enterococcus faecalis | |
thiamine diphosphate | - |
Enterococcus faecalis | |
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 | Comment | Organism |
---|---|---|
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 |
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 |