Literature summary for 3.1.27.8 extracted from

Zhang, Z.; Hao, Z.; Wang, Z.; Li, Q.; Xie, W.
Structure of human endonuclease V as an inosine-specific ribonuclease (2014), Acta Crystallogr. Sect. D, 70, 2286-2294.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Homo sapiens

Crystallization (Comment)

Organism

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

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C225S	site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme	Homo sapiens
C226S	site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme	Homo sapiens
C227S	site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme	Homo sapiens
C228S	site-directed mutagenesis, the mutant shows significantly reduced enzyme activity compared to the wild-type full-length enzyme	Homo sapiens
additional information	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	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, residues Asp52, Glu100 and Asp126 are involved in metal binding	Homo sapiens

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
27800	-	1 * 27800, about, sequence calculation	Homo sapiens
28800	-	recombinant His-tagged enzyme, gel filtration	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Homo sapiens	endonuclease Vs are inosine-specific nucleases that cleave at the second phosphodiester bond 3' to inosine. The human enzyme is most active towards ssRNA but is much less active towards other substrates	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q8N8Q3	-	-

Purification (Commentary)

Purification (Comment)	Organism
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	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
HEK-293T cell	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	endonuclease Vs are inosine-specific nucleases that cleave at the second phosphodiester bond 3' to inosine. The human enzyme is most active towards ssRNA but is much less active towards other substrates	Homo sapiens	?	-	?
additional information	single-stranded deoxyinosine-containing DNA (5'-GCTCGGCTICGGACCGAG-3') and inosine-containing RNA (5'-CUGACUICGGAUCAGGGCC-3') oligonucleotides are synthesized and used as substrates. Substrate-recognition specificity, overview	Homo sapiens	?	-	?

Subunits

Subunits	Comment	Organism
monomer	1 * 27800, about, sequence calculation	Homo sapiens

Synonyms

Synonyms	Comment	Organism
endonuclease V	-	Homo sapiens
EndoV	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Homo sapiens

General Information

General Information	Comment	Organism
evolution	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	Homo sapiens
additional information	structure analysis and substrate-recognition mechanism, overview	Homo sapiens