Information on EC 3.1.27.8 - Ribonuclease V

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.1.27.8
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RECOMMENDED NAME
GeneOntology No.
Ribonuclease V
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of poly(A), forming oligoribonucleotides and ultimately 3'-AMP
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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-
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CAS REGISTRY NUMBER
COMMENTARY hide
74505-36-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
calf
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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the enzyme preserves the general RNase H-like structure, especially in the wedge motif, the metal-binding site and the hypoxanthine-binding pocket. The human enzyme also features several extra insertions and a characteristic four cysteine motif, in which Cys227 and Cys228, two cysteines that are highly conserved in higher eukaryotes, play important roles in catalysis
additional information
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structure analysis and substrate-recognition mechanism, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Poly(A) + H2O
3'-AMP
show the reaction diagram
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Poly(U) + H2O
3'-UMP
show the reaction diagram
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-
-
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rRNA + H2O
?
show the reaction diagram
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-
-
-
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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
rRNA + H2O
?
show the reaction diagram
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-
-
-
-
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required, residues Asp52, Glu100 and Asp126 are involved in metal binding
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0357
Poly(A)
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.014
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
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poly(A)
8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
37 - 38
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poly(A)
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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dermal
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27800
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1 * 27800, about, sequence calculation
28800
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recombinant His-tagged enzyme, gel filtration
52300
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calf, SDS-PAGE
55000
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x * 55000, recombinant N-terminally GST-tagged wild-type and mutant enzymes, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 55000, recombinant N-terminally GST-tagged wild-type and mutant enzymes, SDS-PAGE
monomer
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1 * 27800, about, sequence calculation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged truncated enzyme mutant hEndoV-SF, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.5, 300 mM NaCl, is mixed with 19% PEG 3350, 0.2 M sodium formate, 0.1 M Tris-HCl pH 7.5, or with 1.4 M ammonium sulfate, 0.2 M sodium acetate, 0.1 M Tris-HCl pH 7.5, as crystallization solution, method screening and optimization, 25°C, 2-3 days, X-ray diffraction structure determination and analysis at 2.3 a resolution
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
6 Months, -20°C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, heparin affinity chromatography, and gel filtration
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recombinant N-terminally GST-tagged wild-type and mutant enzymes from Escherichia coli by glutathione affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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recombinant expression of C-terminally FLAG-V5-His6-tagged enzyme in HEK293 cell, recombinant expression of N-terminally GST-tagged wild-type and mutant enzymes in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C225S
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site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme
C226S
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site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme
C227S
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site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme
C228S
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site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme
D52A
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site-directed mutagenesis of an active site residue, inactive mutant
E100A
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site-directed mutagenesis of an active site residue, the mutant shows highly reduced activity compared to the wild-type enzyme
Y91A
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site-directed mutagenesis of an active site residue, the mutant shows highly reduced activity compared to the wild-type enzyme
additional information
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construction of truncated enzyme version representing residues Thr13-Ser250, i.e. hEndoV-SF. The mutant shows reduced enzyme activity compared to the wild-type full-length enzyme