Information on EC 3.1.27.3 - ribonuclease T1

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.1.27.3
-
RECOMMENDED NAME
GeneOntology No.
ribonuclease T1
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
two-stage endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotides ending in Gp with 2',3'-cyclic phosphate intermediates
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
-
-
hydrolysis of phosphoric ester
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
9026-12-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
similar enzyme
-
-
Manually annotated by BRENDA team
strain 7P
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
gene rng
-
-
Manually annotated by BRENDA team
overview
-
-
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
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
adenylyl-3',5'-cytidine
adenosine-2',3'-cyclic phosphate + cytidine
show the reaction diagram
-
ApC
-
-
?
adenylyl-3',5'-cytidine + H2O
adenosine 3'-phosphate + cytidine
show the reaction diagram
ApC + H2O
adenosine 3'-phosphate + cytidine
show the reaction diagram
-
-
-
-
?
GpC + H2O
?
show the reaction diagram
-
enzyme-catalyzed hydrolysis of GpC
-
-
?
GpC + H2O
guanosine 3'-phosphate + cytidine
show the reaction diagram
-
-
-
-
?
GpU + H2O
guanosine 3'-phosphate + uridine
show the reaction diagram
guanosine 2',3'-cyclic phosphate + H2O
guanosine-3'-phosphate
show the reaction diagram
guanosine-2',3'-cyclic phosphate + H2O
guanosine 3'-phosphate
show the reaction diagram
-
hydrolysis
-
-
?
guanylyl(3',5') adenosine + H2O
?
show the reaction diagram
shift in nucleotide conformational equilibrium contributes to increased rate of catalysis of guanylyl(3',5') adenosine 3'-monophosphate versus guanylyl(3',5') adenosine
-
-
?
guanylyl(3',5') adenosine 3'-monophosphate + H2O
?
show the reaction diagram
shift in nucleotide conformational equilibrium contributes to increased rate of catalysis of guanylyl(3',5') adenosine 3'-monophosphate versus guanylyl(3',5') adenosine
-
-
?
guanylyl-3',5'-cytidine
guanosine-2',3'-cyclic phosphate + cytidine
show the reaction diagram
-
Transphosphorylation reaction
-
-
?
guanylyl-3',5'-cytidine + H2O
?
show the reaction diagram
guanylyl-3',5'-cytidine + H2O
guanosine 3'-phosphate + cytidine
show the reaction diagram
-
GpC
-
-
?
guanylyl-3',5'-uridine
guanosine-2',3'-cyclic phosphate + uridine
show the reaction diagram
-
Transphosphorylation reaction with GpU or the diastereomers resulting from thio-substitution of a nonbridging oxygen of GpU, RpGp(S)U and SpGp(S)U as substrates. SpGp(S)U is a very poor substrate for wild type enzyme
-
-
?
guanylyl-3',5'-uridine + H2O
guanosine 3'-phosphate + uridine
show the reaction diagram
-
GpU
-
-
?
poly(I) + H2O
?
show the reaction diagram
-
-
-
-
?
polyinosinic acid + H2O
?
show the reaction diagram
-
polyI
-
-
?
RNA + H2O
?
show the reaction diagram
RNA + H2O
Hydrolyzed RNA
show the reaction diagram
additional information
?
-
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
RNA + H2O
?
show the reaction diagram
-
two-stage endonucleolytic cleavage to 3'-phosphomononucleotides and 3'-phosphooligonucleotides ending in Gp with 2,3'-cyclic phosphate intermediates
-
-
-
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
the enzyme has six binding sites for the cation
H+
-
the cation produces two different tautomers of the histidine residues His53 and His85
Sr2+
-
the enzyme contains a divalent ion-binding site involving Asp49, with preference for this cation. The ion binding at this site stabilizes the protein
additional information
-
Chalaropsis sp., no metal ion requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-guanylic acid
-
substrate analogue
-
3'-guanylic acid
-
substrate analogue
-
3'-N-hydroxyurea-3'-deoxythymidine-5'-phosphate
-
competitive, inhibition is enhanced by nearly 10fold in presence of Zn2 in wild-type enzyme, no enhancement in mutant enzyme E54Q
Acetic anhydride
-
-
barstar
Bromoacetic acid
-
-
Ca2+
-
Neurospora crassa RNase N4
chitosan
Fe3+
-
Aspergillus niger RNase II
Guanylyl-2',5'-guanosine
-
-
histidine
-
-
iodoacetamide
-
iodoacetamide, but not iodoacetate, reacts with these His residues at pH 8.0, but not at pH 5.5
iodoacetate
Mononucleotides
-
e.g. 2'-GMP, 3'-GMP, 5'-GMP, 3'-CMP, 2'(3')-UMP
Ozone
-
loss of activity at pH 7.5, at pH 4.75 the enzyme retains a decreased but distinct enzyme activity towards RNA without alteration of substrate specificity
Phenylglyoxal
phosphate
-
-
Substrate analogs
-
-
-
Tosylglycolate
-
protection by substrate analogs
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
nucleoside 5'-monophosphate
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.021
5'-OHGGGACAGUAUUUG-3'
-
R169K mutant; wild-type
0.00012 - 0.0081
5'-pGGGACAGUAUUUG-3'
0.052 - 4
adenylyl-3',5'-cytidine
0.0268
GpA
-
-
0.069 - 0.9445
GpC
0.0291
GpG
-
-
0.0238
GpU
-
-
1670 - 2010
guanosine-2',3'-cyclic monophosphate
0.0581 - 0.606
guanylyl-3',5'-cytidine
5500 - 10000
guanylyl-3',5'-uridine
0.151 - 0.4
poly(I)
0.1 - 3100
polyinosinic acid
0.321 - 1470
RNA
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
72
5'-OHGGGACAGUAUUUG-3'
Escherichia coli
-
wild-type
126 - 180
5'-pGGGACAGUAUUUG-3'
96
GpA
Aspergillus oryzae
-
-
350
GpC
Aspergillus oryzae
-
-
62
GpG
Aspergillus oryzae
-
-
38
GpU
Aspergillus oryzae
-
-
0.7 - 460
poly(I)
additional information
additional information
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.95 - 1.15
3'-N-hydroxyurea-3'-deoxythymidine-5'-phosphate
0.000033 - 0.00022
chitosan
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2350
purified recombinant enzyme, pH 7.5, 37C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
-
assay at
6
-
Chalaropsis sp.
7.2
-
hydrolysis of guanosine 2',3'-cyclic phosphate
7.3
-
immobilized enzyme
7.4
-
assay at
7.7
-
insolubilized enzyme
7.8
-
native enzyme
8.3
-
cleavage assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 6
-
3: 25% of activity maximum, 6: about 10% of activity maximum, Aspergillus niger RNase II
4 - 9
-
4: 10% (native), 20% (immobilized) of activity maximum, 9: 32% (native), 25% (immobilized) of activity maximum, 10: immobilized enzyme, some activity, native enzyme, inactive
4.5 - 8
-
4.5 and 8: Chalaropsis sp., similar enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
RNA cleavage assay
43
-
immobilized enzyme
45
-
native enzyme
50
-
Aspergillus niger RNase II
65 - 70
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 55
-
20C: 18% of activity maximum, 55C: 15% of activity maximum, inactive at 60C, native enzyme
38 - 80
-
38C: 55% of activity maximum, 80C: 40% of activity maximum, immobilized enzyme
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 4.7
-
calculated from titration curve, 5K mutant
4.9
-
calculated from titration curve, 2K mutant; isoelectric focusing, 2K mutant
5.5
-
isoelectric focusing, 5K mutant
6
-
isoelectric focusing, 3K mutant
6.9
-
calculated from titration curve, 4K mutant; isoelectric focusing, 4K mutant
7.1
-
calculated from titration curve, 3K mutant
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
-
binase is not produced by Bacillus pumilus when bacteria are grown on medium containing an inorganic source of nitrogen. Expression of binase is possible only in Spo0A-OFF cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
in contrast to the wild-type strain in which the inactivated RNase T1-GFP fusion protein is localized at the vacuole only under cold stress or nitrogen starvation, the inactivated RNase T1-GFP fusion protein expressed in the rns4 mutant is localized at the ER and vacuole, both under normal growth conditions and upon ambient stress conditions
Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
-
Neurospora crassa, RNase N4, gel filtration, SDS-PAGE
11090
-
Aspergillus oryzae, amino acid analysis
11840
-
Chalaropsis sp., amino acid composition
12000
-
Chalaropsis sp., low speed equilibrium ultracentrifugation
13000
-
Aspergillus niger, RNase II, gel filtration
53000
-
fusion protein of the antiferritin antibody VL domain and barnase, gel filtration
71100
-
apparent molecular mass of the monomer, determined by sedimentation equilibrium analysis
79000
-
determined by SDS-PAGE, N-terminally His-tagged form
325000
-
tetramer, determined by gel-filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
-
Chalaropsis sp.
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Aspergillus oryzae
-
carboxymethylated RNase T1 crystal structure analysis, PDB ID 1DET
-
crystal structure of RNase T1-T2 (K41E/Y42F/N43R/Y45W/E46N/W59Y), hanging drop vapor diffusion method, 2.1 A resolution
-
hanging drop vapor diffusion method, crystallization of RNase T1 variant RV (mutant RNase T1 K41E/Y42F/N43R/Y45W/E46N)
-
macroseeding
-
RNase T1 crystal structure analysis, PDB ID 9RNT
-
two crystal forms obtained, the classic crystal form I, and the crystals of form II, obtained by repeated seeding
-
the X-ray coordinates of barnase for this study are obtained from the Protein Data Bank, PDB code 1rnb
-
Streptomyces aureofaciens
-
Neurospora crassa RNase N1
-
the crystal structures of RNase NT in complex with either 5'-AMP, 5'-GMP, or 2'-UMP are determined at 1.8 A resolutions by molecular replacement
-
purified recombinant enzyme, hanging drop vapor diffusion method, mixing of 500 nl of 10 mg/mL protein in 20 mM Tris-HCl, pH 7.5, with 500 nl of reservoir solution containing 4 M sodium formate, 0.1 M Bis-Tris propane, pH 7.0, 10% PEG 400, and 300 nl 2 M caesium chloride, 20C, 3 days, X-ray diffraction structure determination and analysis at 1.85 A resolution, molecular replacement method
-
hanging-drop vapor diffusion
-
mutant enzyme Q94K, hanging-drop vapor-diffusion method at 18C
-
RNase Sa-3'IMP complex
-
structures of two crystal forms (I and II) of ribonuclease Sa2 are presented at 1.8 A and 1.5 A resolution. Vapour diffusion from a solution of 1.0% protein by weight in 0.1 M phosphate buffer at pH 7.2 and room temperature with 40% saturated ammonium sulfate as precipitant
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 11
purified recombinant enzyme, stable
729434
2.2
-
37C, 22 h
24201
4 - 10
-
37C, 33 h, immobilized enzyme, highest stability at pH 7, at pH 1.0 and 10.0: about 60% of activity maximum
24202
4.5
-
maximal conformational stability
24208
7
-
-20C, several months, 4C, several weeks, 37C, 22 h
24201
7.5
-
25C, 40 h
24201
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
-
30 min, Neurospora crassa RNase N4
60
-
30 min, 60% loss of activity, Neurospora crassa RNase N4
70
-
30 min, 90% loss of activity, Neurospora crassa RNase N4
90
purified recombinant enzyme, 5 min, over 80% activity remaining
additional information
-
the reversibility of the thermal denaturation is generally between 90% and 95%. Q38A and E74K mutants are slightly more stable than the wild-type enzyme. E41K, E54A and R65A mutants are less stable than the wild-type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Conformational stability
-
Guanidine hydrochloride, pH-dependence of denaturation
-
Immobilized enzyme, heat- and pH-stability
-
Insolubilized enzyme
-
MgCl2 increases conformational stability
-
Na2HPO4 increases conformational stability
-
NaCl and low concentrations of guanidinium chloride stabilize RNase Sa, conformational stability is studied by NMR-monitored hydrogen exchange
NaCl, protects from thermal denaturation, increases transition temperature, suppresses unfolding of enzyme by a denaturant, urea, increases conformational stability
-
Proteases, fairly resistant to proteases: carboxypeptidase A, leucine aminopeptidase, trypsin, chymotrypsin <1>, resistant to trypsin and chymotrypsin, sensitive to pepsin
-
Spermine stabilizes
-
the enzyme is remarkably stable at high salt concentrations
-
Urea, 8 M, stable
-
Urea, pH-dependence of urea denaturation
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
Photooxidation, in presence of Methylene Blue, riboflavine or Rose Bengal inactivation
-
24191
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, pH 7, several months
-
-80C, HEPES buffer, pH 7.5, 0.1 mM EDTA, 1 mM DTT, 50 mM NaCl
-
4C, pH 6, insolubilized enzyme, 5 weeks
-
4C, pH 7, several weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion-exchange chromatography and gel filtration
-
diethylaminoethylcellulose chromatography
-
fusion protein of the antiferritin antibody VL domain and barnase
-
gel filtration
-
ion-exchange chromatography and gel filtration
on porous glass affinity adsorbent
-
recombinant enzyme from Escherichia coli strain BL21(DE3)pLysS to homogeneity by ammonium sulfate fractionation, gel filtration, cation exchange and anion exchange chromatography, and again gel filtration, heparin affinity chromatography, and dialysis
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) pLysS to homogeneity by ammonium sulfate fractionation, gel filtration, cation exchange and anion exchange chromatography, and again gel filtration, heparin affinity chromatography, and dialysis
RNase T1 variants E46N and Y45W/E46N
-
similar enzyme
similar enzyme, partial
using a Super Q Toyopearl column and ion-exchange chromatography on a column of resource Q
-
using Ni-NTA spin columns
-
using Ni2+-nitrilotriacetic acid column chromatography
-
usung a HisTrap HP column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a 1.8 kb DNA fragment, encoding the C-terminal portion of the MycRne polypeptide, residues 332-953, is cloned into p6HisF-11d for expression in Escherichia coli BL21-CodonPlusDE3-RIL cells
-
chemically synthesized gene, mutant Gln58Asp, expressed in Escherichia coli host
-
DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3)pLysS
DNA and amino acid sequence determination and analysis, sequence comparisons, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) pLysS
expression in Epicurian coli
-
expression in Escherichia coli
-
expression in Escherichia coli BL21 carrying plasmid pGEMGX1/ent/Bi as a homogeneous protein
-
expression in Saccharomyces cerevisiae
-
expression of the enzyme in Trp-auxotrophic Escherichia coli, resulting in site-specifically replacement of Trp residues with (2,7-aza)Trp
-
for expression in Escherichia coli BL21DE3-Gold cells
-
fusion of the antiferritin antibody VL domain to barnase and expression in Escherichia coli
-
gene rng, overexpression of wild-type rng and of mutant rngs in rne-1 or rneD1018 alleles mutant strains
-
genetic organization, low level expression of barnase by recombinant Bacillus subtilis strains bearing phoPR-null mutations
-
genetic organization, production of binase by recombinant Bacillus subtilis strains bearing phoPR-null mutations is impossible
-
into the pGEM-T vector for transformation of Escherichia coli JM109 cells, into pPIC9K for expression in Pichia pastoris
-
into the pQE30 vector for expression in Escherichia coli M15 cells
-
overexpression in Neurospora crassa, different promoters are tested, the most promising promoter for recombinant expression is the cfp promoter
overexpression in Saccharomyces cerevisiae. Characterization of an rns4/vps32 mutation in the RNase T1 expression-sensitive strain of Saccharomyces cerevisiae. The rns4 mutant is sensitive to both RNase T1 and ambient stress. In contrast to the wild-type strain in which the inactivated RNase T1-GFP fusion protein is localized at the vacuole only under cold stress or nitrogen starvation, the inactivated RNase T1-GFP fusion protein expressed in the rns4 mutant is localized at the ER and vacuole, both under normal growth conditions and upon ambient stress conditions
-
recombinant enzyme expression in Escherichia coli strain BL21(DE3) pLysS
-
the secondary structure unit mutant S2354 is expressed in Escherichia coli only when His102 is substituted by alanine (H102A)
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
low concentrations of extracellular inorganic phosphate induce the expression of the binase gene
-
upregulation of the KCa channels and the apoptotic p53 genes, downregulation of the antiapoptotic bcl-2 gene under binase action
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E41K
-
with almost the same activity as wild-type enzyme. Glu41 is not involved in substrate binding
E41K/D17K
-
2K mutant
E41K/D17K/D1K
-
3K mutant
E41K/D17K/D1K/D25K
-
4K mutant
E41K/D17K/D1K/D25K/E74K
-
5K mutant
E46N
-
mutant shows an improved ApC/GpC preference with a 1450fold increase in comparison to wild-type activity
E54Q
-
dramatically less active than the wild-type enzyme
E58A
-
less active than the wild type enzyme
E58D
-
with dramatically decreased activity
E74K
-
less active than the wild-type enzyme due to a change in the orientation of the catalytic groups
F100A
-
less active than the wild type enzyme
H40A
-
less active than the wild type enzyme
H85Q
-
dramatically less active than the wild-type enzyme
H92Q
-
less active than the wild type enzyme
K41E/Y42F/N43R/Y45W/E46N/W59Y
-
RNase T1-R2 contains the additional mutation W59Y compared to RNase T1-RV variant. RNase T1-RV is a variant where the specificity is changed from guanine to purine, accompanied with a reduced activity. The additional mutation W59Y increases the activity in comparison to variant RV to 425%
K41M
-
mutant with identical catalytic properties as the wild type enzyme with guanosine-2',3'-cyclic monophosphate as substrate, but less active with polyinosinic acid as substrate
K41T
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mutant with identical catalytic properties as the wild type enzyme with guanosine-2',3'-cyclic monophosphate as substrate, but less active with polyinosinic acid as substrate
N43G
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as active as the wild type enzyme
N43H
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as active as the wild type enzyme
N43I
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as active as the wild type enzyme
N43Q/N44G
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less active than the wild type enzyme
N43T
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as active as the wild type enzyme
N43Y
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as active as the wild type enzyme
N44A
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with dramatically decreased activity
N44H
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less active than the wild type enzyme
Q38A
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less active than the wild-type enzyme
Q46_G47insL
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mutant with a Leu insertion in 47, less active than the wild type enzyme
Q46_N47insQ
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mutant with a Glu insertion in 47, less active than the wild type enzyme
Q46_P47insP
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mutant with a Pro insertion in 47, less active than the wild type enzyme
Q46_P47insQ
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mutant with a Glu insertion in 47, as active as the wild type enzyme
Q46_P47insY
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mutant with a Tyr insertion in 47, less active than the wild type enzyme
Q46_Q47insP
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mutant with a Pro insertion in 47, less active than the wild type enzyme
Q46_R47insR
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mutant with an Arg insertion in 47, less active than the wild type enzyme
Q46_S47insG
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mutant with a Gly insertion in 47, less active than the wild type enzyme
Q46_S47insS
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mutant with a Ser insertion in 47, less active than the wild type enzyme
Q46_T47insS
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mutant with a Ser insertion in 47, less active than the wild type enzyme
R65A
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dramatically less active than the wild-type enzyme
R77K
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with dramatically decreased activity
V16A
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considerably less stable than the wild-type enzyme
V16C
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considerably less stable than the wild-type enzyme
V16S
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considerably less stable than the wild-type enzyme
V16T
-
considerably less stable than the wild-type enzyme
V78A
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considerably less stable than the wild-type enzyme
V78C
-
considerably less stable than the wild-type enzyme
V78S
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considerably less stable than the wild-type enzyme
V78T
-
considerably less stable than the wild-type enzyme
V89A
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considerably less stable than the wild-type enzyme
V89C
-
considerably less stable than the wild-type enzyme
V89S
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considerably less stable than the wild-type enzyme
V89T
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considerably less stable than the wild-type enzyme
Y38F
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less active than the wild type enzyme
Y45S
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as active as the wild type enzyme
Y45W/E46N
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mutant shows an improved ApC/GpC preference with a 2100fold increase in comparison to wild-type activity
D54A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
D54A/E60A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
E60A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
H102A
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the secondary structure unit mutant S2354 is expressed in Escherichia coli only when His102 is substituted by alanine (H102A). The mutant S2354 102A has secondary and tertiary structures and unfolds in a cooperative manner during urea-induced unfolding experiments. S2354H102A interacts with other barnase mutants to hydrolyze RNA
K27A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
K66A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
K66A/D54A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
R59A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
R83Q
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
R87A
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mutant is modeled in silico for gaining insight into modulations of diffusional association behavior, which is reflected in the association rate
D303N
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mutant with amino acid substitution in the binding pocket, inactive enzyme
D346N
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mutant with amino acid substitution in the binding pocket
R169A
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mutant with amino acid substitution in the binding pocket, inactive enzyme
R169K
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mutant with amino acid substitution in the binding pocket
T170V
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mutant with amino acid substitution in the binding pocket
V128A
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mutant with amino acid substitution in the binding pocket
D19N/D22N/E25Q/D31N/D38N/E50Q/E57Q/E76Q/D77N/D79N/E92Q/D93N
site-directed mutagenesis using three different PCR primers
D31N/D38N/E92Q/D93N
site-directed mutagenesis using two different PCR primers
C196T/C603T
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double mutant to avoid restriction enzymatic scission
D1W
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the mutant is studied in concentrated urea and GdnHCl solution with their disulfide bond broken, the results show that long-range effects in a denaturated protein can significantly effect the fluorescence properties
D33A
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Tm-value at pH 7.0 in Mops buffer is 16 C lower than wild-type value. The stability of the mutant enzyme is 6 kcal/mol less than wild-type RNase Sa
D79A
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Tm-value at pH 7.0 in Mops buffer is 9.2 C higher than wild-type value. The stability of the mutant enzyme is 3.3 kcal/mol less than wild-type RNase Sa
D79E
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Tm-value at pH 7.0 in Mops buffer is 0.8 C lower than wild-type value. kcat/Km is identical to wild-type value
D79F
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Tm-value at pH 7.0 in Mops buffer is 9.9 C higher than wild-type value
D79H
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Tm-value at pH 7.0 in Mops buffer is 5.6 C higher than wild-type value
D79I
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Tm-value at pH 7.0 in Mops buffer is 9.6 C higher than wild-type value. kcat/Km is 1.3fold lower than wild-type value
D79K
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Tm-value at pH 7.0 in Mops buffer is 7.6 C higher than wild-type value. kcat/Km is 1.1fold lower than wild-type value
D79L
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Tm-value at pH 7.0 in Mops buffer is 8.7 C higher than wild-type value
D79N
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Tm-value at pH 7.0 in Mops buffer is 5.5 C higher than wild-type value. kcat/Km is 1.1fold lower than wild-type value
D79R
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Tm-value at pH 7.0 in Mops buffer is 9.0 C higher than wild-type value. kcat/Km is 1.1fold higher than wild-type value
D79W
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Tm-value at pH 7.0 in Mops buffer is 7.6 C higher than wild-type value. kcat/Km is 1.2fold lower than wild-type value
D79Y
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Tm-value at pH 7.0 in Mops buffer is 9.6 C higher than wild-type value
Q94K
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Tm-value at pH 7.0 in Mops buffer is 0.8 C higher than wild-type value. Crystal structure shows that the amino group of the Lys forms a hydrogen-bonded ion pair with the carboxyl group of Asp79. The stability of the mutant is about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1 kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged
T76W
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the mutant is studied in concentrated urea and GdnHCl solution with their disulfide bond broken, the results show that long-range effects in a denaturated protein can significantly effect the fluorescence properties
Y52W
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the mutant is studied in concentrated urea and GdnHCl solution with their disulfide bond broken, the results show that long-range effects in a denaturated protein can significantly effect the fluorescence properties
Y55W
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the mutant is studied in concentrated urea and GdnHCl solution with their disulfide bond broken, the results show that long-range effects in a denaturated protein can significantly effect the fluorescence properties
Y81W
-
the mutant is studied in concentrated urea and GdnHCl solution with their disulfide bond broken, the results show that long-range effects in a denaturated protein can significantly effect the fluorescence properties
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
salts induce chain folding of disulfide-reduced and modified RNase T1 into native conformation, enzyme activity is not restored
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the enzyme can be reversibly unfolded and refolded by heating and high concentration of denaturants, such as urea and guanidine hydrochloride, without forming noticeable amounts of aggregates
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine
drug development
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bacterial RNases are promising tools for the development of anticancer drugs. Binase affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs
medicine
additional information
Show AA Sequence (152 entries)
Please use the Sequence Search for a certain query.