Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged wild-type and engineered enzymes in Escherichia coli | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
additional information | in order to increase the peptide linker region length, a 23 amino acid residue polypeptide with a composition of NASSGSSASSPSASNSPGANGSS was inserted between the native interdomain region's Ala and Thr residues. The sequence of the new extended interdomain linker is PANSSIANASSGSSASSPSASNSPGANGSSTLEAE. This peptide sequence is chosen because it has similar structural and dynamic properties to the native interdomain region, and both the designed and the native interdomain linker are predicted to form a flexible and unstructured region. In addition, since DbpA is purified as a native protein, small and polar amino acids, which promote peptide solubility, are placed into the polypeptide insert to discourage the aggregation of extended DbpA and its partition into inclusion bodies. The new interdomain linker is not digested by the Escherichia coli proteolytic enzymes and the extended DbpA is expressed as an intact and soluble protein. Breaking the sequence of the interdomain peptide linker and inserting the 23 amino acids peptide segment causes a decrease in binding affinity, likely as a consequence of formation of non-native interaction between the insert peptide and the RNA molecule or other regions of the protein and not a consequence of disrupting native interactions between the DbpA RNA binding domain and the interdomain linker. The peptide extension is not effecting the formation of the proper ATP pocket, but the ATP turnover rate is affected by the peptide extension. Although the ATP turnover of the extended DbpA is reduced when compared to wild-type DbpA, extended DbpA is a much more efficient enzyme than many members of DEAD-box family of proteins. The reduction on the ATP turnover of the extended DbpA is a consequence of its decrease in binding affinity for RNA. The extension of the interdomain linker region has no effect on the ability of DbpA to perform its helicase function. Thus, the physical connection of DbpA RNA binding domain to the catalytic core is unimportant for the helicase activity of DbpA, suggesting the DbpA protein is a region-specific enzyme, which would unwind any double-helix substrate near hairpin 92 | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics of ATP hydrolysis | Escherichia coli |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
ribosome | - |
Escherichia coli | 5840 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + H2O | Escherichia coli | - |
ADP + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P21693 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His-tagged wild-type and engineered enzymes from Escherichia coli by nickel affinity chromatography and gel filtration | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + H2O | - |
Escherichia coli | ADP + phosphate | - |
? | |
ATP + H2O | the ATP hydrolysis activity of the extended and wild-type DbpA are measured by the pyruvate kinase/lactate dehydrogenase coupled assay. The peptide extension is not effecting the formation of the proper ATP pocket | Escherichia coli | ADP + phosphate | - |
? | |
additional information | the helicase activity of wild-type DbpA and the extended DbpA is investigated by measuring the unwinding of the 5'-32P labeled 9-mer annealed to the unlabeled 32-mer RNA, 32-mer RNA-DNA or the RNA-PEG chimera. DbpA performs RNA structural isomerizations in the ribosome. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core's grasp. The RecA-like domains of the DEAD-box proteins, which form their catalytic core, attack one strand of the RNA double-helix and bend it. The bending process forces the release of the complementary RNA strand. The ATP-binding to the RecA-like domains provides the energy for the single-stranded RNA bending, while the ATP hydrolysis causes the release of the second strand of the double-helix from the catalytic core and the regeneration of the enzymes. The extension of the interdomain linker region has no effect on the ability of DbpA to perform its helicase function. Thus, the physical connection of DbpA RNA binding domain to the catalytic core is unimportant for the helicase activity of DbpA, suggesting the DbpA protein is a region-specific enzyme, which would unwind any double-helix substrate near hairpin 92 | Escherichia coli | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
DbpA | - |
Escherichia coli |
DEAD-box RNA helicase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.72 | - |
ATP | pH 7.5, 22°C, recombinant engineered enzyme | Escherichia coli | |
1.28 | - |
ATP | pH 7.5, 22°C, recombinant wild-type enzyme | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Escherichia coli |
General Information | Comment | Organism |
---|---|---|
malfunction | breaking the sequence of the interdomain peptide linker and inserting the 23 amino acids peptide segment causes a decrease in binding affinity, likely as a consequence of formation of non-native interaction between the insert peptide and the RNA molecule or other regions of the protein and not a consequence of disrupting native interactions between the DbpA RNA binding domain and the interdomain linker. The peptide extension is not effecting the formation of the proper ATP pocket, but the ATP turnover rate is affected by the peptide extension. Although the ATP turnover of the extended DbpA is reduced when compared to wild-type DbpA, extended DbpA is a much more efficient enzyme than many members of DEAD-box family of proteins. The reduction on the ATP turnover of the extended DbpA is a consequence of its decrease in binding affinity for RNA. The extension of the interdomain linker region has no effect on the ability of DbpA to perform its helicase function. Thus, the physical connection of DbpA RNA binding domain to the catalytic core is unimportant for the helicase activity of DbpA, suggesting the DbpA protein is a region-specific enzyme, which would unwind any double-helix substrate near hairpin 92 | Escherichia coli |
additional information | the RecA catalytic core houses DbpA's ATPase and helicase activities. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding | Escherichia coli |
physiological function | DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA performs RNA structural isomerizations in a region of the ribosome that is evolutionarily conserved in all organisms and crucial for their survival | Escherichia coli |