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Literature summary for 2.7.4.16 extracted from
- Characterization of thiamin phosphate kinase in the hyperthermophilic archaeon Pyrobaculum calidifontis (2015), J. Nutr. Sci. Vitaminol., 61, 369-374.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene THI80 or Pcal_0657, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha | Pyrobaculum calidifontis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| R136M | site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type | Pyrobaculum calidifontis |
| S196A | site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type | Pyrobaculum calidifontis |
General Stability
| General Stability | Organism |
|---|---|
| ATP stabilizes the enzyme | Pyrobaculum calidifontis |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| AMP | the mode of AMP inhibition is uncompetitive for both thiamine phosphate and ATP | Pyrobaculum calidifontis |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.000043 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant S196A | Pyrobaculum calidifontis | |
| 0.00013 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged mutant S196A | Pyrobaculum calidifontis | |
| 0.0002 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
| 0.0013 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant R136M | Pyrobaculum calidifontis | |
| 0.039 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
| 0.28 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged mutant R136M | Pyrobaculum calidifontis | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| soluble | - |
Pyrobaculum calidifontis | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Pyrobaculum calidifontis | |
| additional information | no effects of K+ on the enzyme at 0.1-0.5 M | Pyrobaculum calidifontis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + thiamine phosphate | Pyrobaculum calidifontis | - |
ADP + thiamine diphosphate | - |
? | |
| ATP + thiamine phosphate | Pyrobaculum calidifontis JCM 11548 | - |
ADP + thiamine diphosphate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pyrobaculum calidifontis | A3MTW6 | gene Pcal_0657 or THI80 | - |
| Pyrobaculum calidifontis JCM 11548 | A3MTW6 | gene Pcal_0657 or THI80 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by metal affinity chromatography and ultrafiltration | Pyrobaculum calidifontis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + thiamine phosphate | - |
Pyrobaculum calidifontis | ADP + thiamine diphosphate | - |
? | |
| ATP + thiamine phosphate | - |
Pyrobaculum calidifontis JCM 11548 | ADP + thiamine diphosphate | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | SDS-PAGE and gel filtration, neither R136 nor S196 are important for enzyme dimerization | Pyrobaculum calidifontis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| thiamin phosphate kinase | - |
Pyrobaculum calidifontis |
| thiamin pyrophosphokinase | - |
Pyrobaculum calidifontis |
| ThiL | - |
Pyrobaculum calidifontis |
| TP kinase | - |
Pyrobaculum calidifontis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 120 | - |
- |
Pyrobaculum calidifontis |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 80 | 120 | 10% of maximal activity at 80°C, 50% at 100°C, maximal activity at 120°C, inactive on ice | Pyrobaculum calidifontis |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 100 | - |
purified recombinant ThiL protein, loss of about 45% activity after 15 min at 100°C and 20% after 60 min, ATP can partially stabilize the activity, addition of 0.5 M NaCl or 0.5 M KCl to the enzyme solution does not improve the thermostability | Pyrobaculum calidifontis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.006 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant S196A | Pyrobaculum calidifontis | |
| 0.063 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
| 0.092 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant R136M | Pyrobaculum calidifontis | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
- |
Pyrobaculum calidifontis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Pyrobaculum calidifontis | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.53 | - |
AMP | with ATP, pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
| 0.56 | - |
AMP | with thiamine phosphate, pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | biosynthesis pathways of thiamine, thiamine phosphate, and thiamine diphosphate, overview | Pyrobaculum calidifontis |
| additional information | residue Ser196 plays a pivotal role in the catalytic process | Pyrobaculum calidifontis |
| physiological function | in this archaeon, the phosphorylation of thiamine phosphate is the only way to synthesize thiamine diphosphate, it cannot use exogenous thiamine and direct diphosphorylation for the salvage synthesis of thiamine diphosphate | Pyrobaculum calidifontis |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.33 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged mutant R136M | Pyrobaculum calidifontis | |
| 1.6 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
| 46 | - |
ATP | pH 7.0, 100°C, recombinant His-tagged mutant S196A | Pyrobaculum calidifontis | |
| 68 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant R136M | Pyrobaculum calidifontis | |
| 140 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged mutant S196A | Pyrobaculum calidifontis | |
| 320 | - |
thiamine phosphate | pH 7.0, 100°C, recombinant His-tagged wild-type enzyme | Pyrobaculum calidifontis | |
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