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Literature summary for 3.6.1.B17 extracted from
- Novel ATP-independent RNA annealing activity of the Dengue virus NS3 helicase (2012), PLoS One, 7, e36244.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| Single-stranded RNA | viral ATPases, including NS3 from flaviviruses, are stimulated by single-stranded RNA. At RNA saturation, an increase of 13.5fold and 9.6fold is achieved with respect to the basal activities for recombinant mutant NS3-FL and NS3-hel, respectively | Dengue virus |  |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Rosetta pLac | Dengue virus |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| comparison of crystal structures of the full-length NS3 from Dengue virus and Hepatitis C virus indicates a major difference in the relative orientations between the protease and helicase domains in the two proteins. Specifically, a beta-strand in the HCV NS3 clamps the protease domain next to the helicase domain, thereby creating a compact conformational state that differs from the extended conformation observed in the DENV protein | Dengue virus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D284A/E285A | site-directed mutagenesis, inactive mutant showing no ATP hydrolysis. A mutant NS3-hel carrying a two amino acid substitution (D284A-E285A) in the conserved motif II, which corresponds to the Mg2+ co-factor binding loop | Dengue virus |
| H51A | site-directed mutagenesis, the mutation in the protease catalytic site abolishes autoproteolysis of the enzyme | Dengue virus |
| additional information | generation of two recombinant variants of DENV NS3: the first single polypeptide mimics the NS2B-NS3 complex containing the full-length NS3 (618 amino acids) linked to the NS2B hydrophilic region (47 amino acids), this variant named NS3-FL, also carried a mutation in the protease catalytic site (H51A) to avoid autoproteolysis. The second variant NS3-hel represents a truncated NS3, containing the helicase domain (amino acids 171 to 618), ATPase/helicase defective NS3-hel mutant (NS3Amut) conserves the RNA annealing activity | Dengue virus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | the enzyme NS3 establishes an ATP-dependent steady-state between RNA unwinding and strand annealing, Michaelis Menten kinetics | Dengue virus | |
| 0.017 | - |
ATP | mutant NS3-hel, pH 7.0, 30°C | Dengue virus | |
| 0.031 | - |
ATP | mutant NS3-FL, pH 7.0 , 30°C | Dengue virus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Dengue virus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | Dengue virus | - |
ADP + phosphate | - |
? | |
| ATP + H2O | Dengue virus 16681 | - |
ADP + phosphate | - |
? | |
| additional information | Dengue virus | the enzyme preferentially binds single-stranded RNA, while showing low affinity for single or double-stranded DNA (dsDNA) molecules. In addition, enzyme DENV NS3 unwinds RNA duplexes with a 3' to 5' directionality, moving along a tracking RNA strand. The DENV enzyme displays low processivity, unwinds dsDNA molecules inefficiently, and exhibits an RNA triphosphatase activity. In contrast to the requirements for the helicase activity, the RNA annealing activity does not require ATP. Viral enzyme NS3 modulates viral and non-viral RNA structures | ? | - |
? | |
| additional information | Dengue virus 16681 | the enzyme preferentially binds single-stranded RNA, while showing low affinity for single or double-stranded DNA (dsDNA) molecules. In addition, enzyme DENV NS3 unwinds RNA duplexes with a 3' to 5' directionality, moving along a tracking RNA strand. The DENV enzyme displays low processivity, unwinds dsDNA molecules inefficiently, and exhibits an RNA triphosphatase activity. In contrast to the requirements for the helicase activity, the RNA annealing activity does not require ATP. Viral enzyme NS3 modulates viral and non-viral RNA structures | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Dengue virus | O09234 | - |
- |
| Dengue virus 16681 | O09234 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Rosetta pLac by nickel affinity chromatography, dialysis, and ultrafiltration | Dengue virus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | - |
Dengue virus | ADP + phosphate | - |
? | |
| ATP + H2O | best nucleotide substrate | Dengue virus | ADP + phosphate | - |
? | |
| ATP + H2O | - |
Dengue virus 16681 | ADP + phosphate | - |
? | |
| CTP + H2O | - |
Dengue virus | CDP + phosphate | - |
? | |
| GTP + H2O | - |
Dengue virus | GDP + phosphate | - |
? | |
| GTP + H2O | - |
Dengue virus 16681 | GDP + phosphate | - |
? | |
| additional information | the enzyme preferentially binds single-stranded RNA, while showing low affinity for single or double-stranded DNA (dsDNA) molecules. In addition, enzyme DENV NS3 unwinds RNA duplexes with a 3' to 5' directionality, moving along a tracking RNA strand. The DENV enzyme displays low processivity, unwinds dsDNA molecules inefficiently, and exhibits an RNA triphosphatase activity. In contrast to the requirements for the helicase activity, the RNA annealing activity does not require ATP. Viral enzyme NS3 modulates viral and non-viral RNA structures | Dengue virus | ? | - |
? | |
| additional information | enzyme DENV NS3 contains ATP-dependent RNA helicase activity and ATP-independent RNA strand annealing activity, it establishes an ATP-dependent steady-state between RNA unwinding and strand annealing | Dengue virus | ? | - |
? | |
| additional information | the enzyme preferentially binds single-stranded RNA, while showing low affinity for single or double-stranded DNA (dsDNA) molecules. In addition, enzyme DENV NS3 unwinds RNA duplexes with a 3' to 5' directionality, moving along a tracking RNA strand. The DENV enzyme displays low processivity, unwinds dsDNA molecules inefficiently, and exhibits an RNA triphosphatase activity. In contrast to the requirements for the helicase activity, the RNA annealing activity does not require ATP. Viral enzyme NS3 modulates viral and non-viral RNA structures | Dengue virus 16681 | ? | - |
? | |
| additional information | enzyme DENV NS3 contains ATP-dependent RNA helicase activity and ATP-independent RNA strand annealing activity, it establishes an ATP-dependent steady-state between RNA unwinding and strand annealing | Dengue virus 16681 | ? | - |
? | |
| UTP + H2O | - |
Dengue virus | UDP + phosphate | - |
? | |
| UTP + H2O | - |
Dengue virus 16681 | UDP + phosphate | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| ATP-independent RNA annealing activity | - |
Dengue virus |
| DENV helicase | - |
Dengue virus |
| DENV NS3 | - |
Dengue virus |
| nonstructural protein 3 | - |
Dengue virus |
| NS3 | - |
Dengue virus |
| NS3 helicase | - |
Dengue virus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | 30 | assay at | Dengue virus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.2 | - |
ATP | mutant NS3-FL, pH 7.0, 30°C | Dengue virus | |
| 2.8 | - |
ATP | mutant NS3-hel, pH 7.0, 30°C | Dengue virus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
assay at | Dengue virus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | the enzyme NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. Although the helicase domain, comprising amino acids 171 to 618, is an active enzyme, the protease domain has been proposed to have a modulatory role on the helicase and NTPase activities. RNA binding induces a conformational change in the NS3 to a closed form, while no changes in protein structure are observed in different nucleotide-bound states | Dengue virus |
| physiological function | the participation of enzyme NS3, modulating the folding of cis-acting RNA elements by the dual RNA unwinding/annealing activities, provides an additional biological role for this viral protein | Dengue virus |
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