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Literature summary for 2.5.1.21 extracted from
- Biochemical characterization of the water-soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues (2014), FEBS J., 281, 5479-5497.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| DTT | activates, best at 10 mM | Methylococcus capsulatus |  |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene SQS, recombinant expression of soluble His-tagged enzyme in Escherichia coli | Methylococcus capsulatus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D58E | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| D58L | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| D58N | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| D62E | site-directed mutagenesis of the DXXED motif (S1 site), the mutant shows 85% reduced activity compared to the wild-type | Methylococcus capsulatus |
| D62L | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| D62N | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| E61D | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| E61L | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| E61Q | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| R55H | site-directed mutagenesis of the DXXED motif (S1 site), almost inactive mutant | Methylococcus capsulatus |
| R55I | site-directed mutagenesis of the DXXED motif (S1 site), inactive mutant | Methylococcus capsulatus |
| R55K | site-directed mutagenesis of the DXXED motif (S1 site), the mutant shows 96% reduced activity compared to the wild-type | Methylococcus capsulatus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| BPH-652 | a phosphonosulfonate, binding structure | Methylococcus capsulatus | |
| Farnesyl methylenediphosphonate | a substrate analogue, potent enzyme inhibition | Methylococcus capsulatus | |
| additional information | evaluation of the structure of substrate/inhibitor-binding sites via homology modeling, overview. Supplementation of any type of detergent inhibits the enzyme activity during purification | Methylococcus capsulatus | |
| zaragozic acid | competitive type of inhibition | Methylococcus capsulatus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetic analysis | Methylococcus capsulatus | |
| 0.0134 | - |
(2E,6E)-farnesyl diphosphate | pH 8.0, 33°C, recombinant enzyme | Methylococcus capsulatus | |
| 0.095 | - |
NADPH | pH 8.0, 33°C, recombinant enzyme | Methylococcus capsulatus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required, best at 1,25 mM | Methylococcus capsulatus | |
| Mn2+ | activates 40% compared to Mg2+ at 1,25 mM | Methylococcus capsulatus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 (2E,6E)-farnesyl diphosphate + NADPH + H+ | Methylococcus capsulatus | - |
squalene + 2 diphosphate + NADP+ | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Methylococcus capsulatus | Q60AN4 | gene SQS | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant soluble His-tagged enzyme from Escherichia coli by nickel affinity chromatography, supplementation of any type of detergent inhibits the enzyme activity | Methylococcus capsulatus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 (2E,6E)-farnesyl diphosphate + NADPH + H+ | - |
Methylococcus capsulatus | squalene + 2 diphosphate + NADP+ | - |
? | |
| 2 (2E,6E)-farnesyl diphosphate + NADPH + H+ | specific for, the enzyme catalyzes a head-to-head condensation reaction (1'-2,3-linked) between two molecules of farnesyl diphosphate (C15) forms a cyclopropylcarbinyl intermediate, presqualene diphosphate. The subsequent conversion of presqualene diphosphate to squalene involves an extensive rearrangement of the carbon skeleton and an NADPH-dependent reduction reaction | Methylococcus capsulatus | squalene + 2 diphosphate + NADP+ | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| SQS | - |
Methylococcus capsulatus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 33 | - |
assay at | Methylococcus capsulatus |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 10 | 55 | activity range, profil overview | Methylococcus capsulatus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.478 | - |
NADPH | pH 8.0, 33°C, recombinant enzyme | Methylococcus capsulatus | |
| 0.478 | - |
(2E,6E)-farnesyl diphosphate | pH 8.0, 33°C, recombinant enzyme | Methylococcus capsulatus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Methylococcus capsulatus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 10.5 | activity range, profil overview | Methylococcus capsulatus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NADPH | specific for | Methylococcus capsulatus | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | functional analyses of the enzyme's two DXXD(E)D motifs and the highly conserved aromatic amino acid residues, kinetic analysis and reaction mechanism, overview. The potential active-site residues 58DXX61E62D (S1 site) and 213DXX216D217D (S2 site) are assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg2+ ion. The S1 site and the two basic residues R55 and K212 are responsible for the binding of farnesyl diphosphate | Methylococcus capsulatus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 35.05 | - |
(2E,6E)-farnesyl diphosphate | pH 8.0, 33°C, recombinant enzyme | Methylococcus capsulatus | |
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