Application | Comment | Organism |
---|---|---|
synthesis | nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-3-hydroxybutyrate | Streptomyces sp. |
Cloned (Comment) | Organism |
---|---|
gene nphT7, organized in the mevalonate pathway gene cluster, functional expression in Streptomyces albus, overexpression of N-terminally His8-tagged enzyme in Escherichia coli | Streptomyces sp. |
overexpressed in Escherichia coli as an N-terminal His8-tagged protein | Streptomyces sp. |
Protein Variants | Comment | Organism |
---|---|---|
C115A | consumption of malonyl-CoA and an increase in acetyl-CoA. No formation of CoA or acetoacetyl-CoA. C115A mutant enzyme yields acetyl-CoA via its malonyl-CoA decarboxylation activity, presumably by the His256 and Asn286 residues, while it has lost its condensation activity | Streptomyces sp. |
C115A | site-directed mutagenesis, the mutant cleaves malonyl-CoA into acetyl-CoA, but does not form CoA or acetoacetyl-CoA. C115A mutant enzyme yields acetyl-CoA via its malonyl-CoA decarboxylation activity, presumably by the His256 and Asn286 residues, while it has lost its condensation activity | Streptomyces sp. |
H256A | can not ve obtained as a soluble protein | Streptomyces sp. |
H256A | site-directed mutagenesis, the mutant enzyme exhibits acetoacetyl-CoA synthesizing activity, but its specific activity is approximately 40fold lower than that of wild-type NphT7 | Streptomyces sp. |
N286A | this mutant enzyme exhibits detectable acetoacetyl-CoA synthesizing activity, but its specific is approximately 40fold lower than that of wild-type NphT7 | Streptomyces sp. |
N286A | site-directed mutagenesis, the mutant cannot be produced recombionantly as a soluble protein | Streptomyces sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics | Streptomyces sp. | |
0.028 | - |
malonyl-CoA | pH 8.0, 25°C | Streptomyces sp. | |
0.028 | - |
malonyl-CoA | pH 8.0, 30°C, reaction with acetyl-CoA and malonyl-CoA | Streptomyces sp. | |
0.028 | - |
malonyl-CoA | pH 8.0, 30°C, reaction with malonyl-CoA, without acetyl-CoA | Streptomyces sp. | |
0.068 | - |
acetyl-CoA | pH 8.0, 25°C | Streptomyces sp. | |
0.068 | - |
acetyl-CoA | pH 8.0, 30°C, reaction with acetyl-CoA and malonyl-CoA | Streptomyces sp. |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
37000 | - |
2 * 37000, SDS-PAGE | Streptomyces sp. |
37000 | - |
2 * 37000, recombinant N-terminally His8-tagged enzyme, SDS-PAGE | Streptomyces sp. |
63000 | - |
gel filtration | Streptomyces sp. |
63000 | - |
recombinant N-terminally His8-tagged enzyme, gel filtration | Streptomyces sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
acetyl-CoA + malonyl-CoA | Streptomyces sp. | the acetoacetyl-CoA synthesizing enzyme increases the acetoacetyl-CoA supply for mevalonate production | acetoacetyl-CoA + CoA + CO2 | - |
? | |
acetyl-CoA + malonyl-CoA | Streptomyces sp. | - |
CoA + acetoacetyl-CoA + CO2 | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces sp. | - |
- |
- |
Streptomyces sp. | - |
soil-isolated strain, gene nphT7 | - |
Purification (Comment) | Organism |
---|---|
- |
Streptomyces sp. |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
acetyl-CoA + malonyl-CoA = acetoacetyl-CoA + CoA + CO2 | the highly conserved catalytic triad is formed by residues Cys115, His256, and Asn286 in NphT7, reaction mechanism, overview | Streptomyces sp. |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.23 | - |
recombinant mutant H256A, pH 8.0, 25°C | Streptomyces sp. |
8.9 | - |
recombinant His8-tagged wild-type enzyme, pH 8.0, 25°C | Streptomyces sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 malonyl-CoA | - |
Streptomyces sp. | acetoacetyl-CoA + CoA + 2 CO2 | - |
? | |
acetyl-CoA + malonyl-CoA | the acetoacetyl-CoA synthesizing enzyme increases the acetoacetyl-CoA supply for mevalonate production | Streptomyces sp. | acetoacetyl-CoA + CoA + CO2 | - |
? | |
acetyl-CoA + malonyl-CoA | Cys115 in NphT7 functions as a key catalytic residue for the condensation reaction. His256 functions as a catalytic residue for the decarboxylation of the extender substrate malonyl-CoA. No acetoacetyl-CoA synthesizing activity is detected through the condensation of two molecules of acetyl-CoA | Streptomyces sp. | acetoacetyl-CoA + CoA + CO2 | - |
? | |
acetyl-CoA + malonyl-CoA | - |
Streptomyces sp. | CoA + acetoacetyl-CoA + CO2 | - |
ir | |
additional information | the recombinant enzyme catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) results in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity detectable through the condensation of two molecules of acetyl-CoA | Streptomyces sp. | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 37000, SDS-PAGE | Streptomyces sp. |
dimer | 2 * 37000, recombinant N-terminally His8-tagged enzyme, SDS-PAGE | Streptomyces sp. |
Synonyms | Comment | Organism |
---|---|---|
acetoacetyl-coenzyme A synthesizing enzyme | - |
Streptomyces sp. |
More | the enzyme belongs to the thiolase superfamily of enzymes | Streptomyces sp. |
NphT7 | - |
Streptomyces sp. |
thiolase superfamily acetoacetyl-CoA synthase | - |
Streptomyces sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Streptomyces sp. |
30 | - |
assay at | Streptomyces sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Streptomyces sp. |
General Information | Comment | Organism |
---|---|---|
metabolism | the acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily is involved in the mevalonate pathway. Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-3-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms | Streptomyces sp. |