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Literature summary for 3.6.4.13 extracted from

  • Moore, A.F.; Gentry, R.C.; Koculi, E.
    DbpA is a region-specific RNA helicase (2017), Biopolymers, 107, e23001 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.6.4.13

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of N-terminally His-tagged wild-type and engineered enzymes in Escherichia coli Escherichia coli

Protein Variants

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 [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics of ATP hydrolysis Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
ribosome
-
 Escherichia coli 5840
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + H2O Escherichia coli
-
 ADP + phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P21693
-
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
DbpA
-
 Escherichia coli
DEAD-box RNA helicase
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
 assay at Escherichia coli

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Escherichia coli

General Information

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