Information on EC 3.4.21.B57 - pernisine

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.21.B57
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
pernisine
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
the enzyme can digest the pathological prion protein isoform (PrPSc) from different species, e.g. human, bovine, deer and mouse
show the reaction diagram
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human)
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
abnormal prion protein PrP(Sc) + H2O
?
show the reaction diagram
azocasein + H2O
?
show the reaction diagram
Bovine serum albumin + H2O
?
show the reaction diagram
casein + H2O
?
show the reaction diagram
Hemoglobin + H2O
?
show the reaction diagram
N-succinyl-AAPF-4-nitroanilide + H2O
N-succinyl-AAPF + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
N-succinyl-Ala-Ala-Pro-Leu-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Leu + 4-nitroaniline
show the reaction diagram
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24.3% compared to the activity with N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
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-
?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
ovalbumin + H2O
?
show the reaction diagram
pro-Tk-subtilisin + H2O
Tk-subtilisin + propeptide
show the reaction diagram
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autoactivation
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-
?
proform pernisine + H2O
mature pernisine + signal sequence-N-terminal pro-region
show the reaction diagram
the enzyme performs autoproteolytical cleavage of its N-terminal pro-region for activation
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-
?
succinyl-Ala-Ala-Pro-Leu-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Leu + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
Tk-RNase H2 + H2O
?
show the reaction diagram
ribonuclease H2 from Thermococcus kodakarensis, pulse proteolysis using the superstable subtilisin-like serine protease Tk-subtilisin in highly concentrated guanidine hydrochloride to unfold the highly stable substrate protein. The native state of Tk-RNase H2 is completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) is degraded by Tk-subtilisin, identification of the cleavage sites. Structure analysis of unfolded substrate states
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
pro-Tk-subtilisin + H2O
Tk-subtilisin + propeptide
show the reaction diagram
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autoactivation
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-
?
proform pernisine + H2O
mature pernisine + signal sequence-N-terminal pro-region
show the reaction diagram
Q9YFI3
the enzyme performs autoproteolytical cleavage of its N-terminal pro-region for activation
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
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the maximal activity of the purified pernisine seen for 20 mM NaCl in the absence of 1 mM CaCl2 increases to more than 2fold after the addition of CaCl2, with even larger relative enhancement by CaCl2 at higher NaCl concentrations
additional information
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NaCl and CaCl2 together do not show any cumulative effects, as they appear to separately affect the activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
4-hydroxymercuribenzoate
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10 mM, 80% inhibition
AMPHITOL 20Y-B
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presence of 1% (w/v) AMPHITOL 20Y-B causes strong inhibitions, resulting in an enzyme retaining only 50% of its activity
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Aprotinin
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dithiothreitol
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5 mM, 64% loss of activity in absence of CaCl2
DTT
42% and 52% inhibition at 1 mm and 5 mM
Guanidine HCl
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5 mM, 65% loss of activity in absence of CaCl2, activation in presence of 1 mM CaCl2
iodoacetamide
slight inhibition
Soybean trypsin inhibitor
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1 mg/ml, complete inhibition
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Thermococcus kodakaraensis serpin
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irreversibly inhibits more strongly at 80C than at 40C. The covalent inhibitory complex is highly stable and the ester bond between serpin and protease can be hydrolyzed only in a harsh condition, in which most proteases are denatured
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Thermococcus kodakaraensis subtilisin propeptide
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potent noncompetitive inhibitor of the mature domain
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Urea
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5 mM, 50% loss of activity in absence of CaCl2
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
guanidinium hydrochloride
90% activation at 4 M
Urea
slight activation at 1-4 M
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4 - 8
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
26 - 290
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000025
Thermococcus kodakaraensis subtilisin propeptide
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20C, pH 9.5
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
140
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80C, pH 9.5
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
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in the presence of 1 mM CaCl2, at 92C
6.8
native enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 10
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active between pH 3.0 and pH 10.0, in the presence of 1 mM CaCl2, at 92C
3.5 - 8
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active between pH 3.5 and pH 8.0, in absence of CaCl2, at 92C
4.5 - 9.1
the recombinant enzyme shows more than 90% activity within this pH range
6 - 12
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pH 6.0: about 50% of maximal activity, pH 12.0: about 40% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85
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substrate: azocasein, in absence of CaCl2
100
recombinant codon-optimised wild-type enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 120
activity range
40 - 90
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the enzyme exhibits 10% to 20% of the maximal activity at 40C or 90C
58 - 99
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active between 58 C and 99 C
60 - 120
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active in a broad range of temperature, 60C: about 35% of maximal activity, 120C: 80% of maximal activity
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23000
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x * 23000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms
35160
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gel filtration
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
40000
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gel filtration
42000
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gel filtration
43783
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1 * 43783, calculation from sequence
45000
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active-site mutant S255A of pro-Tk-subtilisin
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
63000
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gel filtration, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)
66000
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x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallization of the active-site mutant S255A of pro-Tk-subtilisin. The crystal is grown at 4C by the sitting-drop vapour-diffusion method. Native X-ray diffraction data are collected to 2.3 A resolution.They crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 A. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.6 A(3) Da(-1) and the solvent content was 53.1%
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crystallization of the Pro-S324A/DELTACa6 mutant enzyme using the sitting-drop vapor-diffusion method at 4C
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DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) is crystallized using sitting-drop vapor-diffusion method at 4C. DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) is crystallized using hanging-drop method at 20C. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A. These results suggest that the Ca2+-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)
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sitting-drop vapour-diffusion method at 4C. The crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin), which is refolded in the presence of Ca2+ and absence of Tk-propeptide, is determined at 2.16 A resolution. This structure is the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin
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sitting-drop, vapor-diffusion method at 20 C, the crystal structure of the active-site mutant of the proenzyme lacking C-propeptide (ProN-Tk-S359A) is determined at 2.0 A resolution
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the crystal structure of Pro-F17H/S324A is nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin
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the crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) reveals that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, completely stable
58
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Tm-value of Tk-S359C in absence of CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of CaCl2 and 7.5C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9C in Tm in the absence of Ca2+ and 7.5C in Tm in the presence of Ca2+
58.9
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Tm value of a mutant enzyme without beta-jelly roll domain (Tk-S359A/CDeltaJ), 10 mM CaCl2
70
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20 min, stable in presence of CaCls, about 60% loss of activity in absence of CaCl2
80.1
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Tm-value of Tk-S359C in presence of 10 mM CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of CaCl2 and 7.5C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9C in Tm in the absence of Ca2+ and 7.5C in Tm in the presence of Ca2+
83.9
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Tm-value of ProC-Tk-S359C in absence of CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of CaCl2 and 7.5C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9C in Tm in the absence of Ca2+ and 7.5C in Tm in the presence of Ca2+
87.6
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Tm-value of ProC-Tk-S359C in presence of 10 mM CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of CaCl2 and 7.5C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9C in Tm in the absence of Ca2+ and 7.5C in Tm in the presence of Ca2+
88.3
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Tm value of mutant enzyme S359A, 10 mM CaCl2
additional information
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attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
1 mM CaCl2 stabilizes the enzyme for up to 4 h at 120C
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in the presence of 0.05% (w/v) nonionic surfactants, such as EMULGEN LS-114 or RHEODOL Tw-0120 V, and 0.01% (w/v) EDTA, Tk-SP retains almost its entire initial activity
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in the presence of anionic surfactants, the enzyme is unstable, losing up to 80% of its activity
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in the presence of anionic surfactants, Tk-SP is unstable, losing up to 80% of its activity
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less stable in the presence of QUARTAMIN 60 W
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stable in the presence of 0.1% (w/v) AMPHITOL 20Y-B
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the activity is retained or even enhanced in the presence of nonionic, cationic (except in the presence of SANIZOL C), and amphoteric surfactants at both 0.1 and 1% (w/v)
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the beta-jelly roll domain is required for hyperstabilization of the protein. This domain contains two Ca2+ ions and contributes to the stabilization of the protein only in a Ca2+-bound form
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the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity
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the enzyme is highly stable in the presence of both 0.1 and 1% (w/v) nonionic surfactants
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the enzyme is stabilized in the presence of the Ca2+
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the enzyme retains its activity in the presence of surfactants tested at 80C and 90C
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the enzyme retains more than 100% of its activity in the presence of four of the nonionic surfactants, namely, EMULGEN 147, EMULGEN LS-114, EMULGEN PP-290, and RHEODOL Tw-0120 V. It is less stable in the presence of QUARTAMIN 60 W
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-mercaptoethanol
dithiothreitol
guanidine-HCl
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant proteins, Deltaloop-Tk-subtilisin (Ca2+-binding loop is removed), DeltaCa2-Pro-S324A (Ca2+-binding site Ca2 is removed), and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed), are purified in a Ca2+-bound form
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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
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli BL21(DE3)
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expression in Escherichia coli of pernisine, lacking the leader sequence a fusion protein with glutathione-S-transferase
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expression of wid-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. The enzyme is refolded in a form with a putative prosequence
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overproduction in Escherichia coli of an enzyme derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), its derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the C-propeptide (ProC*)
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overproduction of pro-S255A in Escherichia coli BL21-Codon-Plus(DE3)
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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
the propeptide and mature domain are independently overproduced in Escherichia coli
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Tk-S359A without N- and C-propeptides is overproduced in Escherichia coli in a soluble form
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
to clarify the role of Ca2+ ions (Ca1, Ca6, and Ca7) Pro-TKS derivatives are constructed that lack the Ca1 ion (Pro-TKS/DELTACa1), Ca6 ion (Pro-TKS/DELTACa6), and Ca7 ion (Pro-TKS/DELTACa7), and their active site mutants (Pro-S324A/DELTACa1, Pro-S324A/DELTACa6, and Pro-S324A/DELTACa7, respectively). Pro-TKS/DELTACa6 and Pro-TKS/DELTACa7 fully mature into their active forms upon incubation at 80C for 30 min as do Pro-TKS. The mature enzymes are as active as Tk-subtilisin at 80 C, indicating that the Ca6 and Ca7 ions are not important for activity. Pro-TKS/DELTACa1 matures poorly at 80C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/DELTACa1 is determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives are comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca2+ ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/DELTACa1 and Pro-S324A/DELTACa6 are decreased by 26.6 and 11.7C, respectively, in Tm compared to that of Pro-S324A. The half-lives of Tk-subtilisin/DELTACa6 and Tk-subtilisin/DELTACa7 at 95C are 8fold and 4fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S355A
site-directed mutagenesis, catalytically inactive active site mutant
Pro-Tk-S359C
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construction of an enzyme derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C). Pro-Tk-S359C is purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first
ProC-Tk-S359C
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construction of an enzyme derivative lacking the N-propeptide (ProC-Tk-S359C). The C-propeptide is autoprocessed and degraded when ProC-Tk-S359C is incubated at 80 C in the absence of Ca2+. However, it is not autoprocessed in the presence of Ca2+. The enzymatic activity of ProC-Tk-S359C is higher than (but comparable to) that of Tk-S359C, an enzyme derivatives lacking both propeptides, suggesting that the C-propeptide is not important for activity. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of Ca2+ and 7.5 C in the presence of Ca2+, indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C
S255A
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active-site mutant enzyme
S324C
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site-directed mutagenesis, structure comparison of the mutant pro-enzyme with the wild-type pro-enzyme
S359C
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S359C is more stable than S359A. Tm value of is 58.0C in the presence of 2.5 M GdnHCl and the absence of Ca2+ and 80.1C in the presence of 6 m GdnHCl and 10 mm CaCl2
Tk-S359C
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construction of an enzyme derivative lacking both propeptides (Tk-S359C). The enzymatic activity of ProC-Tk-S359C, an enzyme derivatives lacking the N-propeptide is higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9C in the absence of Ca2+ and 7.5 C in the presence of Ca2+, indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine