Information on EC 3.6.4.B7 - RadA recombinase

Word Map on EC 3.6.4.B7
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.4.B7
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
RadA recombinase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isolated from East Pacific Rise and Mid-Atlantic Ridge deep sea, gene radA
-
-
Manually annotated by BRENDA team
isolated from East Pacific Rise and Mid-Atlantic Ridge deep sea, gene radA
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
RadA intein; gene radA
UniProt
Manually annotated by BRENDA team
RadA intein; gene radA
UniProt
Manually annotated by BRENDA team
gene radA
UniProt
Manually annotated by BRENDA team
gene radA
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O
AMP + diphosphate
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O
AMP + diphosphate
show the reaction diagram
O29269
the enzyme catalyses efficient D-loop formation and strand exchange at temperatures of 60-70C, capable of promoting strand transfer through at least 1200 bp of duplex DNA
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
Mg2+ als well as K+ ions are absorbed at the ATPase center. K+ (but not Na+), stimulates the ATP hydrolysis reaction with an apparent dissociation constant of about 40 mM. The strand exchange activity of the wild-type enzyme is also stimulated by potassium with an apparent dissociation constant of 35 mM
KCl
stimulates the ATPase activity in the absence of ssDNA as well as the strand-exchange activity in the presence of AMPPNP
Mn2+
the enzyme requires the presence of bivalent cations, such as Mg2+ and Mn2+
NaCl
-
20 mM NaCl used in this mixture was found to be optimal for ATP hydrolysis
NH4Cl
stimulates the ATPase activity in the absence of ssDNA as well as the strand-exchange activity in the presence of AMPPNP
RbCl
stimulates the ssDNA-dependent ATPase activity. 1.0 M RbCl does not stimulate the ATPase activity of MmRadA in the absence of DNA
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
stRadC2
ST1830, Sulfolobus tokodaii RadA paralogue stRadC2 is involved in DNA recombination via interaction with recombinase RadA and the Holliday junction Hjc. stRadC2 inhibits the strand exchange activity of RadA and facilitates Hjc-mediated Holliday junction DNA cleavage in vitro. Recombinant expression of His-tagged stRadC2
-
Sulfolobus solfataricus single-stranded DNA binding protein
-
the tetrameric form of Sulfolobus solfataricus single-stranded DNA binding protein significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Direct interaction between Sulfolobus solfataricus single-stranded DNA binding protein and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Rad54 protein
-
the Rad54 protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the SsoRadA, to stimulate DNA strand exchange
-
RadC1
-
Sto0579, enhances the ATPase and strand invasion activities of RadA
-
RadC2
-
Sto1830, interacts with both RadA and Hjc, a Holliday junction resolvase
-
replication protein A
-
stimulates the RadA promoted strand-exchange reaction in vitro
-
ssDNA
SsoRal1 protein
-
in addition to constraining SsoRadA ssDNA-dependent ATPase activity, SsoRal1 enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. This results in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggests a filament stabilization function for the SsoRal1 protein
-
SsoRal3
UniProtID Q97X93, pI of 6.7, a recombinase paralogue from Sulfolobus solfataricus that enhances SsoRadA ssDNA binding and strand displacement. SsoRal3 protein is a ssDNA-dependent ATPase that can catalyze strand invasion at both saturating and subsaturating concentrations. It can bind both ssDNA and dsDNA, but its binding preference is altered by the presence or absence of ATP. SsoRal3 alters SsoRadA ssDNA-dependent ATPase activity, addition of SsoRal3 to SsoRadA nucleoprotein filaments reduces total ATPase activity. Subsaturating concentrations of SsoRal3 increase the ssDNA binding activity of SsoRadA approximately 9fold and also increase the persistence of SsoRadA catalyzed strand invasion products. SsoRal3 functions to stabilize the SsoRadA presynaptic filament. SsoRal3 can catalyze strand invasion and extends the persistence of SsoRadA-mediated D-loops
-
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
49 - 531
ATP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017 - 2.5
ATP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 0.17
ATP
4
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85
the rate of the ATP hydrolysis increases with temperature up to 85C, the maximum temperature at which the protein maintains its secondary structure
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 80
50C: about 40% of maximal activity, 80C: about 60% of maximal activity
65 - 85
-
DNA-dependent ATP hydrolysis occurrs exclusively at elevated temperatures (65C, 75C, 85C), and very little hydrolysis occurrs at 37C
75
the enzyme exhibits a biphasic Arrhenius plot of ATP hydrolysis with two characteristic energies of activation with a break point at 75C. The activation energy below 75C is higher than that above the break point. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75C are greater than those at lower temperatures
80 - 102
-
preformation of presynaptic complex at 80C guarantees the conservation of at least a part of the activity up to 102C. Activity is close to zero at temperatures below 65C
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35867
-
x * 35867, calculated from sequence
38400
-
x * 38400, SDS-PAGE, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oligomer
polymer
undecamer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, PDB ID 1T4G
-
crystal structure analysis, enzyme with bound AMP-PNP, PDB ID 1T4G
-
crystallization of a hexameric form of a truncated Methanococcus voltae RadA protein devoid of its small N-terminal domain, crystals are grown by the hanging drop method The RadA hexamers further assemble into two-ringed assemblies
-
hanging drop crystallization method at 21 C. Enzyme in complex with AMP-PNP and K+ (2.7 A resolution), enzyme in complex with AMP-PNP (2.9 A resolution), enzyme in complex with ADP (2.4 A resolution)
-
hanging drop crystallization method at 21C crystal structure of the enzyme in complex with ADP and Mg2+ at 2.1 A resolution
-
hanging drop crystallization method at room temperature, enzyme in complex with the ATP analog AMP-PNP at 2.0 A resolution. The RadA filament is a 106.7 A pitch helix with six subunits per turn. The DNA binding loops L1 and L2 are located in close proximity to the filament axis. The ATP analog is buried between two RadA subunits
-
crystal structure analysis, PDB ID 1PZN
-
purified His-tagged wild-type RadA intein and engineered minimized PhoRadA intein mutant, sitting drop vapor diffusion, mixing of 100 nl of protein and 100 nl of precipitant solution at 20C, X-ray diffraction structure determination and analysis at 1.58-1.75 A resolution, molecular replacement. Comparison between the NMR and crystal structures of PhoRadA intein
crystal structure analysis, PDB ID 1T4G
-
crystal structure analysis, PDB IDs 2DFL and 2BKE
-
crystal structure of full-length enzyme, quality crystals are grown at 4C using the hanging drop vapour diffusion method against a reservoir containing 0.5 ml 0.1 M TrisHCl, pH 9.5 with 10% (v/v) tert-butanol, resolution of 3.2 A. crystal structure reveals a conformation of fine filaments in the absence of nucleotides
-
crystallized using the hanging drop vapor diffusion method, crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. These five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation
-
expression as a MBP-RadA-His6-fusion protein in Escherichia coli
-
hanging drop vapour diffusion method, crystal structure of the left-handed archaeal RadA helical filament
-
crystal structure analysis, PDB ID 1T4G
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 87
the enzyme maintains its secondary structure and activities in vitro at high temperatures, up to 87C. It also shows high stability of 18.3 kcal/mol at 25C and neutral pH
90
-
prerincubation of 0.015 mM enzyme for 6 min, complete inactivation of the protein
94
purified recombinant His-tagged RadA, remains almost unaffected after 4 h incubation
100
-
after boiling of 0.18 mM enzyme in 20 mM NaCl for 30 min, the enzyme retains one-third of its original activity. Two factors, preliminary presynaptic complex formation and high protein concentration, save, at least partially, the activity of RadADa protein, even at 100C
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
guanidine-HCl
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant proteins K120A and K120R
-
recombinant enzyme from Escherichia coli strain Rosetta by affinity chromatography, dialysis, anion exchange cchromatography
recombinant His-tagged wild-type RadA intein and engineered minimized RadA intein from Escherichia coli strain ER2566 by nickel affinity chromatography, dialysis, His-tag cleavage through yeast ubiquitin-like specific protease, and again nickel affinity chromatography to remove the tag, followed by ultrafiltration
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) using ammonium sulfate fractionation and nickel affinity chromatography, to homogeneity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene radA DNA and amino acid sequence determination and analysis, recombinant overexpression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
gene radA, DNA and amino acid sequence determination and analysis, phylogenetic analysis and genotyping of deep sea isolates using the enzyme sequence, overview
-
gene radA, highly efficient cis-splicing of PhoRadA intein, recombinant expression of His-tagged enzyme in Escherichia coli strain ER2566
gene radA, recombinant expression in Escherichia coli strain Rosetta
hexagonal MmRadA crystals (space group P61) are grown by the hanging-drop vapour-diffusion method and grew to maximum dimensions of 0.1 x 0.1 x 0.3 mm. Crystal structure of enzyme in complex with AMPPNP, crystal structure of the enzyme in complex with AMPPNP and potassium ions and crystal structure of the enzyme in complex with AMPPNP and ammonium ions
overexpressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of radA might be constitutive
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D246A
-
loss of binding a second Mg2+. Initial ATP turnover rate is reduced by about 20-fold
D246N
-
initial ATP turnover rate is reduced by about 20-fold
D302K
-
mutant protein shows comparable strand exchange efficiencies in the presence of either potassium or sodium
E151D
-
mutant protein retains potassium preference in promoting strand exchange. Reduced ATPase activity and normal strand exchange activity
E151K
-
mutant protein retains potassium preference in promoting strand exchange. Reduced ATPase activity and normal strand exchange activity
K120A
-
reduced ATPase activity, mutant K120A is able to bind ssDNA with ATP, ADP, or ATPgammaS under saturating protein conditions, but failed to bind well at subsaturating concentrations with ATP or ATPgammaS
K120R
-
reduced ATPase activity, mutant only binds ATP in the presence of ssDNA
K27A
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, 90100% reduction of the surface plasmon resonance binding signal, exhibits weaker affinity to dsDNA as compared to wild-type protein; mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, mutant exhibits slower ssDNA association rate
K27R
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
K60A
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein; mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, mutants is defective in dsDNA binding
K60R
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, binds dsDNA as well as wild-type protein
R217A
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
R217K
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, mutant exhibits slower ssDNA association rate, surface plasmon resonance binding signals is similar to that of wild-type protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
R223A
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, 90100% reduction of the surface plasmon resonance binding signal, mutants is defective in dsDNA binding
R223K
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, surface plasmon resonance binding signals is similar to that of wild-type protein, mutants is defective in dsDNA binding
R229A
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, 90100% reduction of the surface plasmon resonance binding signal, mutants is defective in dsDNA binding
R229K
-
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, surface plasmon resonance binding signals is similar to that of wild-type protein, mutants is defective in dsDNA binding
K120A
-
reduced ATPase activity, mutant K120A is able to bind ssDNA with ATP, ADP, or ATPgammaS under saturating protein conditions, but failed to bind well at subsaturating concentrations with ATP or ATPgammaS
-
K120R
-
reduced ATPase activity, mutant only binds ATP in the presence of ssDNA
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
diagnostics
molecular biology
pharmacology