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Literature summary for 3.4.21.B57 extracted from
- Codon optimisation is key for pernisine expression in Escherichia coli (2015), PLoS ONE, 10, e0123288.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| guanidinium hydrochloride | 90% activation at 4 M | Aeropyrum pernix |  |
| Urea | slight activation at 1-4 M | Aeropyrum pernix | |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant overexpression of and His-tagged wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised S355A active site mutant (pernisineS355Aco), with or without additional GST-tag or maltose-binding-protein-tag, in Escherichia coli strain BL21(DE3) requires codon preference optimisation and de-novo DNA synthesis. Undetectable expression level of unmodified wild-type enzyme, method evaluation | Aeropyrum pernix |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| S355A | site-directed mutagenesis, catalytically inactive active site mutant | Aeropyrum pernix |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| 2-mercaptoethanol | 49% and 62% inhibition at 1 mm and 5 mM | Aeropyrum pernix | |
| DTT | 42% and 52% inhibition at 1 mm and 5 mM | Aeropyrum pernix | |
| EDTA | - |
Aeropyrum pernix | |
| EGTA | - |
Aeropyrum pernix | |
| iodoacetamide | slight inhibition | Aeropyrum pernix | |
| PMSF | - |
Aeropyrum pernix | |
| SDS | 9% and 90% inhibition at 0.1% and 3% | Aeropyrum pernix | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| extracellular | - |
Aeropyrum pernix | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Ca2+ | required for enzyme stability at higher temperatures, bound Ca2+ increases the thermostability of the subtilases or protects them from autolysis | Aeropyrum pernix | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 36000 | - |
x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE | Aeropyrum pernix |
| 55000 | - |
x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE | Aeropyrum pernix |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | Aeropyrum pernix | enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human) | ? | - |
? | |
| proform pernisine + H2O | Aeropyrum pernix | the enzyme performs autoproteolytical cleavage of its N-terminal pro-region for activation | mature pernisine + signal sequence-N-terminal pro-region | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Aeropyrum pernix | Q9YFI3 | - |
- |
Posttranslational Modification
| Posttranslational Modification | Comment | Organism |
|---|---|---|
| proteolytic modification | the enzyme needs to be heat-activated for 1 h at 90°C in activation buffer containing 10 mM HEPES, 1 mM CaCl2, pH 8.0, through autoproteolytical cleavage of its N-terminal pro-region from the 55 kDa inactive proform to the 36 kDa active form. The cleavage site of the proregion appears to be between Gln92 and Ala93 | Aeropyrum pernix |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged codon-optimised wild-type and codon-optimised S355A active site mutant from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Aeropyrum pernix |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| azocasein + H2O | - |
Aeropyrum pernix | ? | - |
? | |
| casein + H2O | - |
Aeropyrum pernix | ? | - |
? | |
| additional information | enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human) | Aeropyrum pernix | ? | - |
? | |
| proform pernisine + H2O | the enzyme performs autoproteolytical cleavage of its N-terminal pro-region for activation | Aeropyrum pernix | mature pernisine + signal sequence-N-terminal pro-region | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE | Aeropyrum pernix |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| subtilase | - |
Aeropyrum pernix |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 100 | - |
recombinant codon-optimised wild-type enzyme | Aeropyrum pernix |
| 105 | - |
native enzyme | Aeropyrum pernix |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | 120 | activity range | Aeropyrum pernix |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | - |
purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, completely stable | Aeropyrum pernix |
| 80 | - |
purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 20% activity | Aeropyrum pernix |
| 110 | - |
purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 30% activity | Aeropyrum pernix |
| 120 | - |
purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 50% activity | Aeropyrum pernix |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6.8 | - |
native enzyme | Aeropyrum pernix |
| 7 | - |
recombinant codon-optimised wild-type enzyme | Aeropyrum pernix |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 4.5 | 9.1 | the recombinant enzyme shows more than 90% activity within this pH range | Aeropyrum pernix |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | pernisine is a subtilisin-like serine protease (i.e., a subtilase) with the catalytic triad of Asp149, His184 and Ser355. Catalytic nucleophile Ser355 has an important role in pernisine activity, but not in its activation process | Aeropyrum pernix |
| physiological function | enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human) | Aeropyrum pernix |
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