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Literature summary extracted from

  • Cheng, M.; Yoshiyasu, H.; Okano, K.; Ohtake, H.; Honda, K.
    Redirection of the reaction specificity of a thermophilic acetolactate synthase toward acetaldehyde formation (2016), PLoS ONE, 11, e0146146.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.2.1.6 1-methoxy-5-methylphenazinium methyl sulfate
-
 Thermus thermophilus

Application

EC Number Application Comment Organism
2.2.1.6 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
4.1.1.1 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(Commentary)

EC Number Cloned (Comment) Organism
2.2.1.6 expressed in Escherichia coli JM109 cells Thermus thermophilus
2.2.1.6 gene als, recombinant expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and Rosetta 2 (DE3) Thermus thermophilus

Protein Variants

EC Number Protein Variants Comment Organism
2.2.1.6 H474R site-directed mutagenesis Thermus thermophilus
2.2.1.6 H474R the mutant shows reduced activity compared to the wild type enzyme Thermus thermophilus
2.2.1.6 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
2.2.1.6 H747R mutation leads to 3fold increased acetaldehyde formation, with 30% decrease in acetolactate formation Thermus thermophilus
2.2.1.6 K139R site-directed mutagenesis Thermus thermophilus
2.2.1.6 K139R the mutant shows slightly reduced activity compared to the wild type enzyme Thermus thermophilus
2.2.1.6 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
2.2.1.6 V172A site-directed mutagenesis Thermus thermophilus
2.2.1.6 V172A the mutant shows reduced activity compared to the wild type enzyme Thermus thermophilus
2.2.1.6 Y35N site-directed mutagenesis Thermus thermophilus
2.2.1.6 Y35N the mutant shows reduced activity compared to the wild type enzyme Thermus thermophilus
2.2.1.6 Y35N/K139R/V172A/H474R the mutant shows strongly reduced activity compared to the wild type enzyme Thermus thermophilus
2.2.1.6 Y35N/K139R/V172A/H474R shows 3.1fold higher acetaldehyde-forming activity than the wild-type Thermus thermophilus
4.1.1.1 H747R mutation leads to 3fold increased acetaldehyde formation, with 30% decrease in acetolactate formation Thermus thermophilus
4.1.1.1 Y35N/K139R/V172A/H474R shows 3.1fold higher acetaldehyde-forming activity than the wild-type Thermus thermophilus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.2.1.6 Mg2+ required Thermus thermophilus

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 Thermus thermophilus
-
 2-acetolactate + CO2
-
 ?
2.2.1.6 2 pyruvate Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
 2-acetolactate + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.2.1.6 Thermus thermophilus Q5SJ01
-
-
2.2.1.6 Thermus thermophilus Q5SJ01 gene als
-
2.2.1.6 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 Q5SJ01
-
-
2.2.1.6 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 Q5SJ01 gene als
-
4.1.1.1 Thermus thermophilus Q5SJ01 acetolactate synthase, EC 2.2.1.6
-
4.1.1.1 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 Q5SJ01 acetolactate synthase, EC 2.2.1.6
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.2.1.6 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

EC Number Reaction Comment Organism Reaction ID
2.2.1.6 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
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.2.1.6 7.96
-
 mutant Y35N/K139R/V172A/H474R, pH 7.0, 60°C Thermus thermophilus
2.2.1.6 8.13
-
 mutant H747R, pH 7.0, 60°C Thermus thermophilus
2.2.1.6 12.6
-
 wild-type, pH 7.0, 60°C Thermus thermophilus
4.1.1.1 1.45
-
 wild-type, pH 7.0, 60°C Thermus thermophilus
4.1.1.1 4.51
-
 mutant Y35N/K139R/V172A/H474R, pH 7.0, 60°C Thermus thermophilus
4.1.1.1 5.52
-
 mutant H747R, pH 7.0, 60°C Thermus thermophilus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.2.1.6 2 pyruvate
-
 Thermus thermophilus (S)-acetolactate + CO2
-
 ?
2.2.1.6 2 pyruvate
-
 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 (S)-acetolactate + CO2
-
 ?
2.2.1.6 2 pyruvate
-
 Thermus thermophilus 2-acetolactate + CO2
-
 ?
2.2.1.6 2 pyruvate
-
 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 2-acetolactate + CO2
-
 ?
2.2.1.6 additional information non-enzymatic decarboxylation of acetolactate to acetoin Thermus thermophilus ?
-
 ?
2.2.1.6 additional information non-enzymatic decarboxylation of acetolactate to acetoin Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ?
-
 ?
4.1.1.1 pyruvate
-
 Thermus thermophilus acetaldehyde + CO2 in wild-type, about 10% of the acetolactate forming activity ?
4.1.1.1 pyruvate
-
 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 acetaldehyde + CO2 in wild-type, about 10% of the acetolactate forming activity ?

Subunits

EC Number Subunits Comment Organism
2.2.1.6 homotetramer
-
 Thermus thermophilus

Synonyms

EC Number Synonyms Comment Organism
2.2.1.6 ALS
-
 Thermus thermophilus
2.2.1.6 TTHA1213
-
 Thermus thermophilus
4.1.1.1 TTHA1213
-
 Thermus thermophilus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.2.1.6 60
-
 recombinant enzyme in a coupled acetaldehyde-forming assay with enzyme TtALDH Thermus thermophilus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.2.1.6 60
-
 recombinant wild-type enzyme is highly stable up to 100 min, while mutant enzyme loses over 80% activity within 100 min Thermus thermophilus
2.2.1.6 60
-
 the enzyme remains stable after incubation at 60°C for 90min Thermus thermophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.2.1.6 7
-
 acetolactate-forming activity of the recombinant enzyme, and acetaldehyde-forming activity in a coupled assay with enzyme TtALDH Thermus thermophilus

Cofactor

EC Number Cofactor Comment Organism Structure
2.2.1.6 thiamine diphosphate
-
 Thermus thermophilus
2.2.1.6 thiamine diphosphate dependent on, TPP binding site of the model structure of enzyme TtALS, overview Thermus thermophilus
4.1.1.1 thiamine diphosphate
-
 Thermus thermophilus

General Information

EC Number General Information Comment Organism
2.2.1.6 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
2.2.1.6 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
2.2.1.6 additional information structure homology modeling of wild-type enzyme and H474R enzyme mutant, structure comparisons, overview Thermus thermophilus