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Information on EC 4.6.1.20 - ribonuclease U2 and Organism(s) Ustilago sphaerogena and UniProt Accession P00654
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4.6.1.20 ribonuclease U2IUBMB Comments
The enzyme secreted by the fungus Ustilago sphaerogena cleaves at the 3'-phosphate group of purines, and catalyses a two-stage endonucleolytic cleavage. The first reaction produces 5'-hydroxy-phosphooligonucletides and 3'-phosphooligonucleotides ending in Ap or Gp with 2',3'-cyclic phosphodiester, which are released from the enzyme. The enzyme then hydrolyses these cyclic compounds in a second reaction that takes place only when all the susceptible 3',5'-phosphodiester bonds have been cyclised. The second reaction is a reversal of the first reaction using the hydroxyl group of water instead of the 5'-hydroxyl group of ribose. The overall process is that of a phosphorus-oxygen lyase followed by hydrolysis to form the 3'-nucleotides.
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Word Map
- 4.6.1.20
- sphaerogena
- ustilago
-
main-chain
-
deamidation
- succinimide
- alpha-sarcin
- trinucleotides
-
3'-terminal
-
isoaspartate
- analysis
The taxonomic range for the selected organisms is: Ustilago sphaerogena
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
+
=
+
[RNA] containing adenosine
+
=
an [RNA fragment]-3'-adenosine-3'-phosphate
+
a 5'-hydroxy-ribonucleotide-3'-[RNA fragment]
+
=
+
[RNA] containing guanosine
+
=
an [RNA fragment]-3'-guanosine-3'-phosphate
+
a 5'-hydroxy-ribonucleotide-3'-[RNA fragment]
Synonyms
rnase u2, ribonuclease u2, purine-specific rnase, purine-specific ribonuclease, ribonuclease u2a, ribonuclease u2b, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Pleospora RNase
-
-
-
-
Purine specific endoribonuclease
-
-
-
-
Purine-specific ribonuclease
-
-
-
-
Purine-specific RNase
-
-
-
-
Ribonuclease (purine)
-
-
-
-
Ribonuclease U3
-
-
-
-
RNase U2
-
-
-
-
RNase U3
-
-
-
-
Trichoderma koningi RNase III
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of phosphoric ester
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
[RNA]-purine 5'-hydroxy-ribonucleotide-3'-[RNA fragment]-lyase (cyclicizing; [RNA fragment]-3'-purine-nucleoside -2',3'-cyclophosphate-forming and hydrolysing)
The enzyme secreted by the fungus Ustilago sphaerogena cleaves at the 3'-phosphate group of purines, and catalyses a two-stage endonucleolytic cleavage. The first reaction produces 5'-hydroxy-phosphooligonucletides and 3'-phosphooligonucleotides ending in Ap or Gp with 2',3'-cyclic phosphodiester, which are released from the enzyme. The enzyme then hydrolyses these cyclic compounds in a second reaction that takes place only when all the susceptible 3',5'-phosphodiester bonds have been cyclised. The second reaction is a reversal of the first reaction using the hydroxyl group of water instead of the 5'-hydroxyl group of ribose. The overall process is that of a phosphorus-oxygen lyase followed by hydrolysis to form the 3'-nucleotides.
CAS REGISTRY NUMBER
COMMENTARY
37205-57-5
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
-
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
RNA + H2O
?
-
-
-
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
RNA + H2O
3'-Phosphomononucleotides + 3'-phosphooligonucleotides + 2',3'-cyclic nucleotide phosphates
-
r, preferential cleavage at Ap or Gp
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
trinucleotides, synthetic substrates
-
additional information
additional information
-
trinucleotides, synthetic substrates
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
specific activity increases 78fold after purification from crude extracts
additional information
-
specific activity increases 78fold after purification from crude extracts
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
Asn32 of RNase U2A rapidly deaminates and isomerizes in alkaline conditions, conformational mechanism, overview. In solution, the region around Asn32-Gly33 is likely to be in equilibrium between multiple conformers with deamination of Asn32 proceeding when the region adopts an extended conformation
additional information
-
Asn32 of RNase U2A rapidly deaminates and isomerizes in alkaline conditions, conformational mechanism, overview. In solution, the region around Asn32-Gly33 is likely to be in equilibrium between multiple conformers with deamination of Asn32 proceeding when the region adopts an extended conformation
additional information
formation of isoAsp32 induces a single turn unfolding of the alpha-helix from Asp29 to Asp34, and the region from Asp29 to Arg35 forms a U-shaped loop structure, overview. IsoAsp32 protrudes from the surface of the protein, and the abnormal beta-peptide bond in the main-chain and alpha-carboxylate in the side-chain is fully exposed. The structure suggests that the deamidation of the Asn and the isoAsp formation in proteins could confer immunogenicity
additional information
-
formation of isoAsp32 induces a single turn unfolding of the alpha-helix from Asp29 to Asp34, and the region from Asp29 to Arg35 forms a U-shaped loop structure, overview. IsoAsp32 protrudes from the surface of the protein, and the abnormal beta-peptide bond in the main-chain and alpha-carboxylate in the side-chain is fully exposed. The structure suggests that the deamidation of the Asn and the isoAsp formation in proteins could confer immunogenicity
additional information
-
mechanism of isoaspartate formation within the Asp45-Glu46 sequence of ribonuclease U2, overview. In the succinimide-formation reaction water 219 receives a proton from the N atom of Glu46 as a general base and waters 190 and 220 stabilize the tetrahedral intermediate, and in the succinimide-hydrolysis reaction water 219 provides a proton for the N atom of Glu46 as a general acid
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). RNU2_USTSP
114
0
12387
Swiss-Prot
other Location (Reliability: 1), Secretory Pathway (Reliability: 4)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
11000
x * 11000, SDS-PAGE of nonreduced protein, x * 22000, SDS-PAGE of reduced protein
13330
mutant H101Q, MALDI-TOF, posttranslational modification suggested
22000
x * 11000, SDS-PAGE of nonreduced protein, x * 22000, SDS-PAGE of reduced protein
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 11000, SDS-PAGE of nonreduced protein, x * 22000, SDS-PAGE of reduced protein
additional information
additional information
protein does not bind SDS under SDS-PAGE conditions. Electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass, while migration of the reduced protein would be in accordance with the expected molecular mass of the dimer
additional information
formation of isoAsp32 induces a single turn unfolding of the alpha-helix from Asp29 to Asp34, and the region from Asp29 to Arg35 forms a U-shaped loop structure, overview
additional information
-
formation of isoAsp32 induces a single turn unfolding of the alpha-helix from Asp29 to Asp34, and the region from Asp29 to Arg35 forms a U-shaped loop structure, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
isoAsp containing ribonuclease U2B, hanging drop vapor diffusion method, 0.004 ml of 10 mg/ml protein in 50 mM sodium citrate, pH 4.5, containing 50 mM sodium chloride, are mixed with 0.004 ml of reservoir solution containing 0.84 M sodium dihydrogen phosphate and 1.26 M dipotassium hydrogen phosphate, pH 6.9, equilibration against 0.4 ml reservoir solution, 20°C, 2 days, X-ray diffraction structure determination and analysis at 1.32 A resolution
RNase U2A in complex with 2'-adenylic acid and Ca2+ ions, X-ray diffraction structure determination and analysis at 1.03 A resolution
ribonuclease U2 complexed with adenosine 3'-monophosphate, hanging drop vapour diffusion method, 0.005 ml of protein solution containing 20 mg/ml RNase U2A and 8 mM adenosine 3'-monophosphate, pH 4.5, is mixed with 0.005 ml of reservoir solution containing 12.5% w/w PEG 8000, 200 mM calcium acetate, 100 mM sodium cacodylate, pH 3.75, 12.5% w/w PEG 8000, equilibration against 0.4 ml reservoir solution, 20°C, 3 days, X-ray diffraction structure determination and analysis at 0.96-0.99 A resolution
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E49A
mutant with non-specific ribonuclease activity and a tendency to undercut RNA
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native RNase U2A by anion-exchange and 5'-adenylate aminohexyl affinity chromatography
purification of recombinant enzyme using several chromatographic steps, including affinity chromatography on 2´-5'-ADP-Sepharose
purification of recombinant enzyme using several chromatographic steps, including DEAE-cellulose and affinity chromatography on 2'-5'-ADP-Sepharose
native RNase U2A by anion-exchange and 5'-adenylate aminohexyl affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Pichia pastoris, different signal-peptide cleavage processing sites for different recombinant proteins
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
improvement of RNA modification mapping sequence coverage by LC-MS. The non-specific ribonuclease activity RNase U2 E49A substitution mutant provides increased sequence coverage of substrate RNA during modification mapping. Data analysis software is modified to account for non-specific digestion. This combination allows efficient and accurate RNA modification mapping
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Uchida, T.; Egami, F.
Microbial ribonucleases with special reference to RNases T1, T2, N1, and U2
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
4
205-250
1971
Ustilago sphaerogena
-
Yasuda, T.; Inoue, Y.
Studies of catalysis by ribonuclease U2. Steady-state kinetics for transphosphorylation of oligonucleotide and synthetic substrates
Biochemistry
21
364-369
1982
Ustilago sphaerogena
Uchida, T.; Shibata, Y.
An affinity adsorbent, 5-adenylate-aminohexyl-sepharose. I. Purification and properties of two forms of RNase U2
J. Biochem.
90
463-471
1981
Ustilago sphaerogena
Minato, S.; Hirai, A.
Characterization of Ustilago Ribonuclease U2. Effects of chemical modification at glutamic acid-61 and cystine-1 and of organic solvents on the enzymatic activity
J. Biochem.
85
327-334
1979
Ustilago sphaerogena
Uchida, T.; Machida, C.
The specificity for dinucleoside monophosphates of RNase U2 and urea-treated RNase U2
Nucleic Acids Res.
1 Suppl 2
409-413
1978
Ustilago sphaerogena
-
Sato, S.; Uchida, T.
Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena
J. Biochem.
77
795-800
1975
Ustilago sphaerogena
Sato, S.; Uchida, T.
On the interaction of ribonuclease U-2 and substrate analogues
Biochim. Biophys. Acta
383
168-177
1975
Ustilago sphaerogena
Kioke, T.; Uchida, T.; Egami, F.
Synthesis of adenylyl-(3,5)-nucleosides, adenylyl-(3,5)-guanosine 2,3-cyclic phosphate, and oligoadenylic acids by ribonuclease U2
J. Biochem.
69
111-117
1971
Ustilago sphaerogena
Glitz, D.G.; Dekker, C.A.
A ribonuclease from Ustilago sphaerogena. I. Purification and properties of the enzyme
Biochemistry
3
1391-1399
1964
Ustilago sphaerogena
Glitz, D.G.; Dekker, C.A.
A ribonuclease from Ustilago sphaerogena. II. Specificity of the enzyme
Biochemistry
3
1399-1406
1964
Ustilago sphaerogena
Martinez-Ruiz, A.; Garcia-Ortega, L.; Kao, R.; Onaderra, M.; Mancheno, J.M.; Davies, J.; Martinez del Pozo, A.; Gavilanes, J.G.
Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein
FEMS Microbiol. Lett.
189
165-169
2000
Ustilago sphaerogena (P00654), Ustilago sphaerogena, Ustilago sphaerogena CBS 534.71 (P00654)
Martinez-Ruiz, A.; Garcia-Ortega, L.; Kao, R.; Lacadena, J.; Onaderra, M.; Mancheno, J.M.; Davies, J.; Del Pozo, A.M.; Gavilanes, J.G.
RNase U2 and alpha-sarcin: a study of relationships
Methods Enzymol.
341
335-351
2001
Ustilago sphaerogena (P00654)
Garcia-Ortega, L.; De los Rios, V.; Martinez-Ruiz, A.; Onaderra, M.; Lacadena, J.; Martinez del Pozo, A.; Gavilanes, J.G.
Anomalous electrophoretic behavior of a very acidic protein: ribonuclease U2
Electrophoresis
26
3407-3413
2005
Ustilago sphaerogena (P00654)
Alvarez-Garcia, E.; Garcia-Ortega, L.; De los Rios, V.; Gavilanes, J.G.; Martinez-del-Pozo, A.
Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris
Protein Expr. Purif.
65
223-229
2009
Ustilago sphaerogena (P00654), Ustilago sphaerogena
Noguchi, S.
Isomerization mechanism of aspartate to isoaspartate implied by structures of Ustilago sphaerogena ribonuclease U2 complexed with adenosine 3'-monophosphate
Acta Crystallogr. Sect. D
66
843-849
2010
Ustilago sphaerogena
Noguchi, S.
Structural changes induced by the deamidation and isomerization of asparagine revealed by the crystal structure of Ustilago sphaerogena ribonuclease U2B
Biopolymers
93
1003-1010
2010
Ustilago sphaerogena (P00654), Ustilago sphaerogena
Noguchi, S.
Conformational variation revealed by the crystal structure of RNase U2A complexed with Ca ion and 2'-adenylic acid at 1.03 A resolution
Protein Pept. Lett.
17
1559-1561
2010
Ustilago sphaerogena (P00654), Ustilago sphaerogena
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