Activating Compound | Comment | Organism | Structure |
---|---|---|---|
1-methoxy-5-methylphenazinium methyl sulfate | - |
Thermus thermophilus |
Application | Comment | Organism |
---|---|---|
biotechnology | the reaction specificity of acetolactate synthase from Thermus thermophilus can be redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Quadruple mutant Y35N/K139R/V172A/H474R shows 3.1fold higher acetaldehyde-forming activity than the wild-type mainly because of H474R amino acid substitution, which likely generates two new hydrogen bonds near the thiamine diphosphate-binding site | Thermus thermophilus |
Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli JM109 cells | Thermus thermophilus |
gene als, recombinant expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and Rosetta 2 (DE3) | Thermus thermophilus |
Protein Variants | Comment | Organism |
---|---|---|
H474R | site-directed mutagenesis | Thermus thermophilus |
H474R | the mutant shows reduced activity compared to the wild type enzyme | Thermus thermophilus |
H474R | site-directed mutagenesis, the reaction specificity of acetolactate synthase from Thermus thermophilus is redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. The mutation likely generates two new hydrogen bonds near the thiamine diphosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme | Thermus thermophilus |
H747R | mutation leads to 3fold increased acetaldehyde formation, with 30% decrease in acetolactate formation | Thermus thermophilus |
K139R | site-directed mutagenesis | Thermus thermophilus |
K139R | the mutant shows slightly reduced activity compared to the wild type enzyme | Thermus thermophilus |
additional information | genotyping by random mutagenesis, error-prone PCR and mutant library screening leading to the identification of a quadruple mutant with 3.1fold higher acetaldehyde-forming activity than the wild-type, mutant reaction-specificity profiles | Thermus thermophilus |
V172A | site-directed mutagenesis | Thermus thermophilus |
V172A | the mutant shows reduced activity compared to the wild type enzyme | Thermus thermophilus |
Y35N | site-directed mutagenesis | Thermus thermophilus |
Y35N | the mutant shows reduced activity compared to the wild type enzyme | Thermus thermophilus |
Y35N/K139R/V172A/H474R | the mutant shows strongly reduced activity compared to the wild type enzyme | Thermus thermophilus |
Y35N/K139R/V172A/H474R | shows 3.1fold higher acetaldehyde-forming activity than the wild-type | Thermus thermophilus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Thermus thermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 pyruvate | Thermus thermophilus | - |
2-acetolactate + CO2 | - |
? | |
2 pyruvate | Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | - |
2-acetolactate + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus thermophilus | Q5SJ01 | - |
- |
Thermus thermophilus | Q5SJ01 | gene als | - |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | Q5SJ01 | - |
- |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | Q5SJ01 | gene als | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strains JM109 and Rosetta 2 (DE3) partially by heat treatment at 50°C or 70°C for 30 min | Thermus thermophilus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 pyruvate = 2-acetolactate + CO2 | the reaction proceeds via the formation of a common substrate-cofactor-enzyme complex. The carbonyl addition of pyruvate to thiamine diphosphate yields a predecarboxylation intermediate followed by the elimination of carbon dioxide, resulting in the formation of a central and highly reactive intermediate, 2-hydroxyethyl-thiamine diphosphate. The carboligation between 2-hydroxyethyl-TPP and the second pyruvate molecule leads to the liberation of the reaction product, acetolactate, and the catalytic cycle is completed | Thermus thermophilus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
7.96 | - |
mutant Y35N/K139R/V172A/H474R, pH 7.0, 60°C | Thermus thermophilus |
8.13 | - |
mutant H747R, pH 7.0, 60°C | Thermus thermophilus |
12.6 | - |
wild-type, pH 7.0, 60°C | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 pyruvate | - |
Thermus thermophilus | (S)-acetolactate + CO2 | - |
? | |
2 pyruvate | - |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | (S)-acetolactate + CO2 | - |
? | |
2 pyruvate | - |
Thermus thermophilus | 2-acetolactate + CO2 | - |
? | |
2 pyruvate | - |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | 2-acetolactate + CO2 | - |
? | |
additional information | non-enzymatic decarboxylation of acetolactate to acetoin | Thermus thermophilus | ? | - |
? | |
additional information | non-enzymatic decarboxylation of acetolactate to acetoin | Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | - |
Thermus thermophilus |
Synonyms | Comment | Organism |
---|---|---|
ALS | - |
Thermus thermophilus |
TTHA1213 | - |
Thermus thermophilus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
recombinant enzyme in a coupled acetaldehyde-forming assay with enzyme TtALDH | Thermus thermophilus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
recombinant wild-type enzyme is highly stable up to 100 min, while mutant enzyme loses over 80% activity within 100 min | Thermus thermophilus |
60 | - |
the enzyme remains stable after incubation at 60°C for 90min | Thermus thermophilus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
acetolactate-forming activity of the recombinant enzyme, and acetaldehyde-forming activity in a coupled assay with enzyme TtALDH | Thermus thermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | - |
Thermus thermophilus | |
thiamine diphosphate | dependent on, TPP binding site of the model structure of enzyme TtALS, overview | Thermus thermophilus |
General Information | Comment | Organism |
---|---|---|
evolution | acetolactate synthase and pyruvate decarboxylase are both thiamine diphosphate-dependent enzymes that use pyruvate as a substrate, but they produce different products.Whereas pyruvate decarboxylase catalyzes the non-oxidative decarboxylation of pyruvate to acetaldehyde, acetolactate synthase, which is involved in the biosynthesis of branched amino acids (Val, Leu, Ile), catalyzes the carboligation between two pyruvate molecules to form an acetolactate molecule and carbon dioxide, structural and functional similarities of the enzymes, overview | Thermus thermophilus |
metabolism | acetolactate synthase is a thiamine diphosphate-dependent enzyme that is involved in the biosynthesis of branched amino acids (Val, Leu, Ile), catalyzing the carboligation between two pyruvate molecules to form an acetolactate molecule and carbon dioxide | Thermus thermophilus |
additional information | structure homology modeling of wild-type enzyme and H474R enzyme mutant, structure comparisons, overview | Thermus thermophilus |