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Literature summary extracted from
- In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa (2008), J. Am. Chem. Soc., 130, 4954-4967.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 2.1.1.324 | expressed in Escherichia coli BL21(DE3) cells | Saccharopolyspora spinosa |
| 2.6.1.110 | expressed in Escherichia coli BL21(DE3) cells | Saccharopolyspora spinosa |
| 4.2.1.164 | expression in Escherichia coli BL21 | Saccharopolyspora spinosa |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 4.2.1.164 | 0.049 | - |
dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose | pH 7.5, 24°C | Saccharopolyspora spinosa |  |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 2.1.1.324 | Mg2+ | 2 mM used in assay conditions | Saccharopolyspora spinosa | |
| 4.2.1.164 | iron-sulfur centre | the enzyme contains 1.2 irons per monomer as determined by ferrozine quantitation, consistent with the enzyme having a [2Fe-2S] cluster | Saccharopolyspora spinosa | |
Molecular Weight [Da]
| EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|---|
| 1.1.1.384 | 37732 | - |
1 * 38000, SDS-PAGE, 1 * 37732, calculated, His-tagged recombinant protein | Saccharopolyspora spinosa |
| 1.1.1.384 | 38000 | - |
gel filtration | Saccharopolyspora spinosa |
| 2.1.1.324 | 28000 | - |
2 * 28000, SDS-PAGE | Saccharopolyspora spinosa |
| 2.1.1.324 | 28730 | - |
2 * 28730, calculated from amino acid sequence | Saccharopolyspora spinosa |
| 2.1.1.324 | 61200 | - |
gel filtration | Saccharopolyspora spinosa |
| 2.6.1.110 | 43000 | - |
2 * 43000, SDS-PAGE | Saccharopolyspora spinosa |
| 2.6.1.110 | 43356 | - |
2 * 43356, calculated from amino acid sequence | Saccharopolyspora spinosa |
| 2.6.1.110 | 85900 | - |
gel filtration | Saccharopolyspora spinosa |
| 4.2.1.164 | 51000 | - |
2 * 51000, SDS-PAGE | Saccharopolyspora spinosa |
| 4.2.1.164 | 109400 | - |
- |
Saccharopolyspora spinosa |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.1.1.384 | TDP-3,4-didehydro-2,6-dideoxy-alpha-D-glucose + NADPH + H+ | Saccharopolyspora spinosa | - |
TDP-4-dehydro-2,6-dideoxy-alpha-D-glucose + NADP+ | - |
? | |
| 2.1.1.324 | S-adenosyl-L-methionine + dTDP-4-amino-2,3,4,6-tetradeoxy-alpha-D-erythro-hexopyranose | Saccharopolyspora spinosa | - |
S-adenosyl-L-homocysteine + dTDP-alpha-D-forosamine | - |
? | |
| 2.6.1.110 | TDP-4-dehydro-2,3,6-trideoxy-D-glucose + 2-oxoglutarate | Saccharopolyspora spinosa | - |
TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose + L-glutamate | - |
r | |
| 4.2.1.164 | dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ | Saccharopolyspora spinosa | the enzyme is involved in TDP-D-forosamine biosynthesis in the spinosyn pathway | dTDP-4-dehydro-2,3,6-trideoxy-alpha-D-hexopyranose + H2O + 2 oxidized ferredoxin [iron-sulfur] cluster | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.1.1.384 | Saccharopolyspora spinosa | Q9ALN5 | - |
- |
| 2.1.1.324 | Saccharopolyspora spinosa | Q9ALP0 | - |
- |
| 2.6.1.110 | Saccharopolyspora spinosa | - |
- |
- |
| 4.2.1.164 | Saccharopolyspora spinosa | Q9ALN8 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 2.1.1.324 | Ni-NTA column chromatography and Sephacryl S-200 gel filtration | Saccharopolyspora spinosa |
| 2.6.1.110 | Ni-NTA column chromatography and Sephacryl S-200 gel filtration | Saccharopolyspora spinosa |
| 4.2.1.164 | - |
Saccharopolyspora spinosa |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.1.1.384 | TDP-3,4-didehydro-2,6-dideoxy-alpha-D-glucose + NADPH + H+ | - |
Saccharopolyspora spinosa | TDP-4-dehydro-2,6-dideoxy-alpha-D-glucose + NADP+ | - |
? | |
| 2.1.1.324 | S-adenosyl-L-methionine + dTDP-4-amino-2,3,4,6-tetradeoxy-alpha-D-erythro-hexopyranose | - |
Saccharopolyspora spinosa | S-adenosyl-L-homocysteine + dTDP-alpha-D-forosamine | - |
? | |
| 2.6.1.110 | dTDP-2,6-dideoxy-alpha-D-threo-hexopyranos-4-ulose + L-glutamate | in the presence of pyridoxal 5'-phosphate | Saccharopolyspora spinosa | dTDP-4-amino-2,4,6-trideoxy-alpha-D-arabino-hexopyranose + 2-oxoglutarate | - |
ir | |
| 2.6.1.110 | dTMP-5-amino-2,6-anhydro-1,3,4,5,7-pentadeoxy-1-phosphono-D-arabino-heptitol + 2-oxoglutarate | in the presence of pyridoxal 5'-phosphate | Saccharopolyspora spinosa | dTMP-2,6-anhydro-5-dehydro-1,3,4,7-tetradeoxy-1-phosphono-D-arabino-heptitol + L-glutamate | - |
r | |
| 2.6.1.110 | TDP-4-dehydro-2,3,6-trideoxy-D-glucose + 2-oxoglutarate | - |
Saccharopolyspora spinosa | TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose + L-glutamate | - |
r | |
| 2.6.1.110 | TDP-4-dehydro-2,6-dideoxy-D-glucose + 2-oxoglutarate | - |
Saccharopolyspora spinosa | TDP-4-amino-2,4,6-trideoxy-D-glucose + L-glutamate | - |
r | |
| 2.6.1.110 | TDP-C-4-amino-2,3,4,6-tetradeoxy-alpha-D-erythro-hexopyranose + 2-oxoglutarate | - |
Saccharopolyspora spinosa | ? | - |
? | |
| 4.2.1.164 | dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ | the enzyme is involved in TDP-D-forosamine biosynthesis in the spinosyn pathway | Saccharopolyspora spinosa | dTDP-4-dehydro-2,3,6-trideoxy-alpha-D-hexopyranose + H2O + 2 oxidized ferredoxin [iron-sulfur] cluster | - |
? | |
| 4.2.1.164 | dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ | the enzyme is capable of catalyzing C-3 deoxygenation in the presence of dithionite or the reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase. Conversion is significantly more efficient using reductase pairs than using dithionite. In the absence of an electron source and in the presence of L-glutamate, SpnQ catalyzes a transamination reaction, converting dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose to TDP-4-amino-2,4,6-trideoxy-D-glucose | Saccharopolyspora spinosa | dTDP-4-dehydro-2,3,6-trideoxy-alpha-D-hexopyranose + H2O + 2 oxidized ferredoxin [iron-sulfur] cluster | - |
? | |
| 4.2.1.164 | additional information | TDP-4-dehydro-6-deoxy-D-glucose is not a substrate | Saccharopolyspora spinosa | ? | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.1.1.384 | monomer | 1 * 38000, SDS-PAGE, 1 * 37732, calculated, His-tagged recombinant protein | Saccharopolyspora spinosa |
| 2.1.1.324 | homodimer | 2 * 28000, SDS-PAGE | Saccharopolyspora spinosa |
| 2.1.1.324 | homodimer | 2 * 28730, calculated from amino acid sequence | Saccharopolyspora spinosa |
| 2.6.1.110 | homodimer | 2 * 43000, SDS-PAGE | Saccharopolyspora spinosa |
| 2.6.1.110 | homodimer | 2 * 43356, calculated from amino acid sequence | Saccharopolyspora spinosa |
| 4.2.1.164 | dimer | 2 * 51000, SDS-PAGE | Saccharopolyspora spinosa |
| 4.2.1.164 | dimer | 2 * 50361, calculated from sequence | Saccharopolyspora spinosa |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.1.1.384 | SpnN | - |
Saccharopolyspora spinosa |
| 2.1.1.324 | SpnS | - |
Saccharopolyspora spinosa |
| 2.6.1.110 | SpnR | - |
Saccharopolyspora spinosa |
| 2.6.1.110 | TDP-4-keto-2,3,6-trideoxy-D-glucose 4-aminotransferase | - |
Saccharopolyspora spinosa |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 4.2.1.164 | 0.043 | - |
dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose | pH 7.5, 24°C | Saccharopolyspora spinosa | |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.164 | 7.5 | - |
assay at | Saccharopolyspora spinosa |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.1.1.384 | NADH | NADPH is preferred over NADH | Saccharopolyspora spinosa | |
| 1.1.1.384 | NADPH | preferred over NADH | Saccharopolyspora spinosa | |
| 2.6.1.110 | pyridoxal 5'-phosphate | - |
Saccharopolyspora spinosa | |
| 4.2.1.164 | iron-sulfur centre | the enzyme contains 1.2 irons per monomer as determined by ferrozine quantitation, consistent with the enzyme having a [2Fe-2S] cluster | Saccharopolyspora spinosa | |
| 4.2.1.164 | pyridoxal 5'-phosphate | the enzyme is dependent on pyridoxal 5'-phosphate | Saccharopolyspora spinosa | |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.1.1.384 | physiological function | enzyme is involved in insecticide spinosyn synthesis. It acts sequentially with 2,3-dehydratase SpnO to catalyze C-2 deoxygenation of TDP-4-dehydro-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-dehydro-2,6-dideoxy-D-glucose | Saccharopolyspora spinosa |
| 4.2.1.164 | metabolism | the enzyme is involved in TDP-D-forosamine biosynthesis in the spinosyn pathway | Saccharopolyspora spinosa |
kcat/KM [mM/s]
| EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 4.2.1.164 | 0.88 | - |
dTDP-4-dehydro-2,6-dideoxy-alpha-D-glucose | pH 7.5, 24°C | Saccharopolyspora spinosa | |
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