Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 5.6.2.4 extracted from

  • Windgassen, T.A.; Keck, J.L.
    An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase (2016), Nucleic Acids Res., 44, 9745-9757 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.6.2.4

Cloned(Commentary)

Cloned (Comment) Organism
gene priA, recombinant expression of His-tagged wild-type and mutant PriA enzymes in Escherichia coli strain Rosetta2, recombinant expression of His-tagged PriA incorporated with 4-benzoyl-L-phenylalanine (Bpa) in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
K230A site-directed mutagenesis, a Walker A mutant, mutation of a lysine from motif I that is essential for ATPase activity, inactive mutant, used as negative control Escherichia coli
K328A site-directed mutagenesis, 3-4fold reduced kcat compared to wild-type Escherichia coli
additional information construction of PriA incorporated with 4-benzoyl-L-phenylalanine (Bpa), the priA open reading frame is subcloned from pET15-EcPriA into pBAD/His B vector and subjected to site-directed mutagenesis to substitute an amber stop codon (TAG) at specific EcPriA codons targeted for Bpa substitution Escherichia coli
Q329A site-directed mutagenesis, 2fold reduced kcat compared to wild-type Escherichia coli
Q330A site-directed mutagenesis, 3-4fold reduced kcat compared to wild-type Escherichia coli
R334A site-directed mutagenesis, 3-4fold reduced kcat compared to wild-type Escherichia coli
W333A site-directed mutagenesis, 12fold reduced kcat compared to wild-type Escherichia coli
Y335A site-directed mutagenesis, 14fold reduced kcat compared to wild-type Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
EDTA
-
 Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.000038
-
 ATP recombinant mutant K333A, pH 8.0, 25°C Escherichia coli
0.000068
-
 ATP recombinant mutant K335A, pH 8.0, 25°C Escherichia coli
0.000091
-
 ATP recombinant mutant K330A, pH 8.0, 25°C Escherichia coli
0.0001
-
 ATP recombinant wild-type enzyme, pH 8.0, 25°C Escherichia coli
0.00012
-
 ATP recombinant mutant K329A, pH 8.0, 25°C Escherichia coli
0.00013
-
 ATP recombinant mutant K334A, pH 8.0, 25°C Escherichia coli
0.00014
-
 ATP recombinant mutant K328A, pH 8.0, 25°C Escherichia coli

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 an aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase ADP + phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P17888
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant PriA enzymes from Escherichia coli strain Rosetta2 by nickel affinity and ion exchange chromatography, gel filtration, and dialysis, recombinant His-tagged PriA incorporated with 4-benzoyl-L-phenylalanine (Bpa) from Escherichia coli strain BL21(DE3) by nickel affinity and ion exchange chromatography, and dialysis Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O
-
 Escherichia coli ADP + phosphate
-
 ?
ATP + H2O an aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase Escherichia coli ADP + phosphate
-
 ?
additional information usage of a four-stranded DNA substrate Escherichia coli ?
-
-

Synonyms

Synonyms Comment Organism
PriA
-
 Escherichia coli
PriA DNA helicase
-
 Escherichia coli
SF2 DNA helicase
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 ATPase assay at Escherichia coli
37
-
 helicase assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.03
-
 ATP below, recombinant mutant K230A, pH 8.0, 25°C Escherichia coli
0.15
-
 ATP recombinant mutant K335A, pH 8.0, 25°C Escherichia coli
0.18
-
 ATP recombinant mutant K333A, pH 8.0, 25°C Escherichia coli
0.48
-
 ATP recombinant mutant K328A, pH 8.0, 25°C Escherichia coli
0.53
-
 ATP recombinant mutant K334A, pH 8.0, 25°C Escherichia coli
0.78
-
 ATP recombinant mutant K330A, pH 8.0, 25°C Escherichia coli
1.07
-
 ATP recombinant mutant K329A, pH 8.0, 25°C Escherichia coli
2.17
-
 ATP recombinant wild-type enzyme, pH 8.0, 25°C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 helicase and ATPase assays at Escherichia coli

General Information

General Information Comment Organism
additional information protein-DNA crosslinking defines strand-specific interactions for the PriA ARL, 3'BD and helicase core with a DNA replication fork substrate. PriA-DNA fork crosslinking maps strand- and residue-specific interactions. Modeling of PriA domains predicted to bind to specific DNA regions of an abandoned DNA fork, with location of Bpa incorporation, overview. Reproducible crosslinks to the four-stranded DNA substrate are observed with D17Bpa, Q329Bpa (low level), Q330Bpa, E331Bpa and E492Bpa Escherichia coli
physiological function helicases couple ATP hydrolysis to nucleic acid binding and unwinding Escherichia coli