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 (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 (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 | 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 | 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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | Q04789 | - |
- |
Bacillus subtilis PY79 | Q04789 | - |
- |
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 | 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 | 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 | Comment | Organism |
---|---|---|
AlsS | - |
Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
- |
Bacillus subtilis |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | 65 | activity range, overview | Bacillus subtilis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
- |
Bacillus subtilis |
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 | 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 | 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 |