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Literature summary for 3.6.1.74 extracted from
- Identification of a novel function of the alphavirus capping apparatus. RNA 5-triphosphatase activity of Nsp2 (2000), J. Biol. Chem., 275, 17281-17287 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene nsP2, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Semliki forest virus |
| gene nsP2, recombinant expression of N-terminally His-tagged wild-type enzyme in Escherichia coli strain BL21(DE3) | Sindbis virus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| K192N | site-directed mutagenesis, the mutation in the nucleotide-binding site completely abolishes RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N (N-terminal fragment) | Semliki forest virus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | capping apparatus kinetics, overview | Semliki forest virus | |
| 0.00299 | - |
a 5'-triphospho-[mRNA] | Nsp2-N (N-terminal fragment), pH 7.0, 25°C | Semliki forest virus |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | best at 1.0-2.0 mM | Semliki forest virus |  |
| Mn2+ | best at 0.1 mM, sharp optimum | Semliki forest virus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| a 5'-triphospho-[mRNA] + H2O | Semliki forest virus | - |
a 5'-diphospho-[mRNA] + phosphate | - |
? | |
| a 5'-triphospho-[mRNA] + H2O | Sindbis virus | - |
a 5'-diphospho-[mRNA] + phosphate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Semliki forest virus | P08411 | a polyprotein; SFV, an alphavirus | - |
| Sindbis virus | P03317 | a polyprotein; an alphavirus | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Semliki forest virus |
| recombinant N-terminally His-tagged wild-type enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Sindbis virus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| a 5'-triphospho-[mRNA] + H2O | - |
Semliki forest virus | a 5'-diphospho-[mRNA] + phosphate | - |
? | |
| a 5'-triphospho-[mRNA] + H2O | - |
Sindbis virus | a 5'-diphospho-[mRNA] + phosphate | - |
? | |
| additional information | nonstructural protein Nsp2 of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5'-end of RNA. The same activity is demonstrated for the N-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The C-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) has no RNA triphosphatase activity | Semliki forest virus | ? | - |
- |
|
| additional information | nonstructural protein Nsp2 of Sindbis virus specifically cleaves the gamma,beta-triphosphate bond at the 5'-end of RNA | Sindbis virus | ? | - |
- |
|
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| nonstructural protein 2 | - |
Semliki forest virus |
| nonstructural protein 2 | - |
Sindbis virus |
| nsP2 | - |
Semliki forest virus |
| nsP2 | - |
Sindbis virus |
| RNA 5'-triphosphatase | - |
Semliki forest virus |
| RNA 5'-triphosphatase | - |
Sindbis virus |
| RNA triphosphatase | - |
Semliki forest virus |
| RNA triphosphatase | - |
Sindbis virus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
- |
Semliki forest virus |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 20 | 42 | optimal activity at 25°C, inactivation at 42°C, recombinant enzyme | Semliki forest virus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.092 | - |
a 5'-triphospho-[mRNA] | Nsp2-N (N-terminal fragment), pH 7.0, 25°C | Semliki forest virus |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | 8 | broad optimum | Semliki forest virus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | enzyme Nsp2 has several distinct functions. It has nucleoside triphosphatase (NTPase) activity at its N-terminal half, which is vital for the virus replication. Nsp2 has RNA helicase activity, which utilizes NTP hydrolysis as the energy source. The C-terminal part of the protein is a papain-like protease responsible for the autocatalytic cleavages of the nonstructural polyprotein. The C-terminal part has a nuclear localization sequence, which is responsible for sequestering of about half of the molecules to the nucleus during infection. Furthermore, Nsp2 regulates transcription of the subgenomic 26 S RNA. Nsp2 has yet an additional activity required for capping of the virus mRNAs. Capping of cellular mRNAs occurs in the nucleus and comprises four different reactions. RNA 5'-triphosphatase removes the gamma-phosphate from the 5'-end of the nascent RNA molecule (pppRNA -> ppRNA). Guanylyltransferase reacts with a GTP molecule to form a covalent complex with GMP, which is then transferred from guanylyltransferase to the 5'-end of RNA to form G(5')ppp(5')NpRNA. Methylation by guanine-7N-methyltransferase yields an RNA molecule with the cap0 structure (m7GpppNpRNA). Further methylation by nucleoside-2'-O-methyltransferase of the riboses of the penultimate and the adjacent nucleotides yields cap1 and cap2 structures, respectively. Unlike host cellular mRNAs, the capping of alphavirus RNAs takes place in the cytoplasm and is carried out by reactions that differ from the nuclear reactions. Nsp2 hydrolyzes only the gamma,beta-triphosphate bond at the 5'-end of RNA | Semliki forest virus |
| physiological function | enzyme Nsp2 has several distinct functions. It has nucleoside triphosphatase (NTPase) activity at its N-terminal half, which is vital for the virus replication. Nsp2 has RNA helicase activity, which utilizes NTP hydrolysis as the energy source. The C-terminal part of the protein is a papain-like protease responsible for the autocatalytic cleavages of the nonstructural polyprotein. The C-terminal part has a nuclear localization sequence, which is responsible for sequestering of about half of the molecules to the nucleus during infection. Furthermore, Nsp2 regulates transcription of the subgenomic 26 S RNA. Nsp2 has yet an additional activity required for capping of the virus mRNAs. Capping of cellular mRNAs occurs in the nucleus and comprises four different reactions. RNA 5'-triphosphatase removes the gamma-phosphate from the 5'-end of the nascent RNA molecule (pppRNA -> ppRNA). Guanylyltransferase reacts with a GTP molecule to form a covalent complex with GMP, which is then transferred from guanylyltransferase to the 5'-end of RNA to form G(5')ppp(5')NpRNA. Methylation by guanine-7N-methyltransferase yields an RNA molecule with the cap0 structure (m7GpppNpRNA). Further methylation by nucleoside-2'-O-methyltransferase of the riboses of the penultimate and the adjacent nucleotides yields cap1 and cap2 structures, respectively. Unlike host cellular mRNAs, the capping of alphavirus RNAs takes place in the cytoplasm and is carried out by reactions that differ from the nuclear reactions | Sindbis virus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 30.77 | - |
a 5'-triphospho-[mRNA] | Nsp2-N (N-terminal fragment), pH 7.0, 25°C | Semliki forest virus |
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