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Literature summary for 2.2.1.6 extracted from

  • Sommer, B.; von Moeller, H.; Haack, M.; Qoura, F.; Langner, C.; Bourenkov, G.; Garbe, D.; Loll, B.; Brueck, T.
    Detailed structure-function correlations of Bacillus subtilis acetolactate synthase (2015), ChemBioChem, 16, 110-118.
    View publication on PubMed

Application

Application Comment Organism
synthesis Bacillus subtilis acetolactate synthase can act as key biocatalyst in the formation of isobutanol which is deemed to be a next-generation biofuel and a renewable platform chemical. The enzyme AlsS catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol Bacillus subtilis

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) pLys Express and HMS174(DE3) cells Bacillus subtilis
phylogenetic analysis, recombinant expression of wild-type and mutant C-terminally His-tagged enzymes in Escherichia coli Bacillus subtilis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified enzyme in the presence of thiamine diphosphate and Mg2+, and in a transition state with a 2-lactyl moiety bound to thiamine diphosphate, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement Bacillus subtilis
sitting drop vapor diffusion method, using polyethylene glycol 300 (30%, w/v), CaAc (200 mM), and sodium cacodylate (100 mM, pH 6.5) Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
K40H site-directed mutagenesis, the half-life of the mutant at 50°C is 44 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows reduced activity compared to the wild-type Bacillus subtilis
K40I site-directed mutagenesis, the half-life of the mutant at 50°C is 89 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows highly reduced activity compared to the wild-type Bacillus subtilis
K40I the mutant shows slightly improved activity towards 2-oxoisovalerate compared to the wild type enzyme Bacillus subtilis
K40Y site-directed mutagenesis, the half-life of the mutant at 50°C is 110 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows reduced activity compared to the wild-type Bacillus subtilis
M483N site-directed mutagenesis, the mutant is inactivated at 50°C Bacillus subtilis
additional information structure-guided mutagenesis strategy to generate enzyme AlsS variants Bacillus subtilis
P87A site-directed mutagenesis, the half-life of the mutant at 50°C is 33 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows reduced activity compared to the wild-type Bacillus subtilis
Q124S site-directed mutagenesis, the half-life of the mutant at 50°C is 42 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows reduced activity compared to the wild-type Bacillus subtilis
Q424S site-directed mutagenesis, the half-life of the mutant at 50°C is 104 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows increased activity compared to the wild-type Bacillus subtilis
Q424S/Q487S site-directed mutagenesis, the half-life of the mutant at 50°C is 94 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows highly reduced activity compared to the wild-type Bacillus subtilis
Q487S site-directed mutagenesis, the half-life of the mutant at 50°C is 22 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows reduced activity compared to the wild-type Bacillus subtilis
Q487S the mutant shows wild type activity towards 2-oxoisovalerate Bacillus subtilis
T84V site-directed mutagenesis, the half-life of the mutant at 50°C is 2.5 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows highly reduced activity compared to the wild-type Bacillus subtilis
Y481A site-directed mutagenesis, the half-life of the mutant at 50°C is 19 h, compared to 81 h for the wild-type enzyme, the mutant enzyme shows highly reduced activity compared to the wild-type Bacillus subtilis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Bacillus subtilis
Mg2+ required, the Mg2+ cation is coordinated to residues of a single monomer, binding site structure, overview Bacillus subtilis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2 pyruvate Bacillus subtilis
-
2-acetolactate + CO2
-
?
2 pyruvate Bacillus subtilis PY79
-
2-acetolactate + CO2
-
?
2-oxoisovalerate Bacillus subtilis
-
isobutyraldehyde + CO2
-
?
2-oxoisovalerate Bacillus subtilis PY79
-
isobutyraldehyde + CO2
-
?
additional information Bacillus subtilis enzyme AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg2+ as cofactors. The enzyme also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol ?
-
?
additional information Bacillus subtilis PY79 enzyme AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg2+ as cofactors. The enzyme also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol ?
-
?

Organic Solvent Stability

Organic Solvent Comment Organism
Ethanol the enzyme retains 50% activity in the presence of 13% (v/v) ethanol Bacillus subtilis
isobutanol the enzyme retains 50% activity in the presence of 3% (v/v) isobutanol Bacillus subtilis
n-Butanol the enzyme retains 50% activity in the presence of 3% (v/v) n-butanol Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis Q04789
-
-
Bacillus subtilis PY79 Q04789
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni2+-NTA column chromatography, HiTrap Q column chromatography, and Superdex S200 gel filtration Bacillus subtilis
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli Bacillus subtilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 pyruvate
-
Bacillus subtilis 2-acetolactate + CO2
-
?
2 pyruvate
-
Bacillus subtilis PY79 2-acetolactate + CO2
-
?
2-oxoisovalerate
-
Bacillus subtilis isobutyraldehyde + CO2
-
?
2-oxoisovalerate
-
Bacillus subtilis PY79 isobutyraldehyde + CO2
-
?
additional information enzyme AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg2+ as cofactors. The enzyme also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol Bacillus subtilis ?
-
?
additional information enzyme AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg2+ as cofactors. The enzyme also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol Bacillus subtilis PY79 ?
-
?

Subunits

Subunits Comment Organism
homotetramer the enzyme is a homotetramer formed by dimers of dimers, each monomer is composed of three domains. The alpha-domain (up to N181) is connected by a random coil to the central beta-domain (P195 to A346). The C-terminal gamma-domain (from H376) is connected to the central beta-domain by an alpha-helix and a random coil linker, structure-function analysis of the enzyme, overview. The 12 C-terminal resolved residues of AlsS (D556-K567) fold into a short alpha-helix Bacillus subtilis

Synonyms

Synonyms Comment Organism
AlsS
-
Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
Bacillus subtilis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 65 activity range, overview Bacillus subtilis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
half-life of recombinant C-terminally His-tagged enzyme is 81 h, comparison with half-lives of mutant enzymes, overview Bacillus subtilis
50 60 at 50°C and 60°C, the enzyme shows a half-life of 81 h and 16 h, respectively Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
-
Bacillus subtilis

pH Range

pH Minimum pH Maximum Comment Organism
5 9 activity range, overview Bacillus subtilis
5 7 more than 50% activity between pH 5.0 and 7.0 Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
thiamine diphosphate
-
Bacillus subtilis
thiamine diphosphate dependent on, the thiamine function of ThDP interacts with residues of one monomer and the adjacent monomer Bacillus subtilis

General Information

General Information Comment Organism
evolution the enzyme belongs to the ALS enzyme family that forms a distinct subgroup of ThDP-dependent enzymes. The ALS subfamily differs significantly in structure and possibly in catalytic mechanism, phylogenetic analysis. The ThDP-dependent enzymes cluster into three distinct sequence groups: acetolactate synthases, acetohydroxyacid synthases, and carboxylases. Eventhough ALS and AHAS catalyze the same reaction, they show different cofactors and domain structure: AHAS family enzymes have both catalytic and regulatory subunits, structure comparisons, overview Bacillus subtilis
additional information active site structure, catalytically relevant structure-function relationships, overview Bacillus subtilis