Information on EC 3.1.21.2 - deoxyribonuclease IV

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.1.21.2
-
RECOMMENDED NAME
GeneOntology No.
deoxyribonuclease IV
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endonucleolytic cleavage of ssDNA at apurinic/apyrimidinic sites to 5'-phosphooligonucleotide end-products
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric diester
-
hydrolysis of phosphoric ester
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
37211-67-9
-
58591-37-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene nfo
-
-
Manually annotated by BRENDA team
B.
-
-
Manually annotated by BRENDA team
gene sco4631
-
-
Manually annotated by BRENDA team
gene sco4631
-
-
Manually annotated by BRENDA team
-
Uniprot
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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
21-mer C-Grec + H2O
?
show the reaction diagram
5'-end-labeled 19 nucleotide single-stranded DNA + H2O
?
show the reaction diagram
5'-end-labeled 29 nucleotide double-stranded DNA + H2O
?
show the reaction diagram
double stranded DNA + H2O
?
show the reaction diagram
-
the enzyme prefers to remove mismatched 3'-terminal nucleotides from 3'-recessed, nicked, and gapped double stranded DNA
-
-
?
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
double-stranded native DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides, the enzyme makes predominantly single-strand break, and a few double-strand breaks, the ratio is 3.7:1
-
-
ir
double-stranded nativeDNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
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
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
double-stranded nativeDNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
stimulates activity
CaCl2
-
8% of the activity found compared to optimal Mg2+ concentration
Co2+
-
required for activity
CoCl2
-
can substitute for MgCl2, optimal concentration: 0.01 M, activity 27% higher than at optimal MgCl2 concentration
MgCl2
-
absolute requirement, no activity in absence, optimal concentration: 0.01 M
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
0.05 M
4-Chloromercuriphenylsulfonate
-
-
8-hydroxyquinoline
-
inhibits cleavage of alkylated dsDNA
diethyldithiocarbamic acid
-
-
dithiothreitol
complete inhibition at 50 mM
NaCl
-
0.05 M
NH4Cl
-
0.05 M
p-hydroxymercuribenzoate
-
-
potassium phosphate buffer
-
pH 8.3
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
slight activation
bovine serum albumin
-
130% activity at 1% at pH 8.0 and 37C
-
additional information
-
the enzyme is induced by phage T4, no activation by S-adenosyl-L-methionine, ATP, or both
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000025 - 0.000028
21-mer C Grec
0.00000691
5'-end-labeled 29 nucleotide double-stranded DNA
at pH 8.0 and 37C
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0003 - 0.28
21-mer C Grec
0.0193
5'-end-labeled 29 nucleotide double-stranded DNA
Mycobacterium tuberculosis
P9WQ13
at pH 8.0 and 37C
-
additional information
additional information
Enterobacteria phage T4
-
effects of dC-flanking sequences of the substrate on kinetic parameters of Endo IV
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 8
21-mer C Grec
2790
5'-end-labeled 29 nucleotide double-stranded DNA
Mycobacterium tuberculosis
P9WQ13
at pH 8.0 and 37C
197714
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
relative activity in several Escherichia coli wild-type and mutant strains, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
assay on the uracil-processing activity and 3'-5' exonuclease activity; DNA cleavage assay
7.6
-
assay at
8.4 - 9.2
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38
-
assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Geobacillus kaustophilus (strain HTA426)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
determined by SDS-PAGE
additional information
-
sucrose density gradient sedimentation equilibrium analysis
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
-
4 * 16000
additional information
-
EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. Monomer structure and topology, quarternary structure, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant E118A of EndoII is crystallized in space group P21 with four monomers in the asymmetric unit, with a bound phosphate ion in one of the four active sites of EndoII likely mimicing the scissile phosphate in a true substrate complex. Crystallization of the selenomethionine-substituted E118A mutant, X-ray diffraction structure determination and analysis at 1.9-2.3 A resolution, molecular replacement
-
wild-type enzyme and mutant H69A, hanging-drop vapor diffusion method, mxing of 0.0015 ml of protein solution containing 10 mg/ml protein with 0.002 ml of reservoir solution containing 35% PEG 400 and 100 mM HEPES, pH 5.5, 20C, 7 days, X-ray diffraction structure determination and analysis at 1.55 A resolution
-
hanging drop vapor diffusion method, using 100 mM Tris-HCl, pH 9.0, 4 mM dithiothreitol, 1.5% saturated MgSO4, 16% (w/v) ethylene glycol, 20% (w/v) polyethylene glycol monomethyl ether 2000
using the sitting-drop vapor-diffusion method
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
loss of 41% activity after 30 min, loss of 92% activity after 2 h, with addition 50% glycerol only 6% activity is lost within 2 h
45
-
10 min, fraction IV, loss of 10% activity
60
-
10 min, fraction IV, complete inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glycerol and bovine serum albumin stabilize
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, purified enzyme, 40 mM potassium phosphate, pH 6.5, or 50 mM Tris-HCl, pH 8.0, with 0.1 mM DTT or 2-mercaptoethanol, moderately stable
-
-20C, Tris-buffer, pH 6.8, 50 mM NaCl, 0.5 mM DTT, 50% glycerol
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
GST-Endo IV fusion protein
-
GST-Endo IV fusion proteins: wild-type and mutants W88R and S176N
-
phage T4 infected
-
Streptactin column chromatography, Resource Q column chromatography, and Superdex 200 gel filtration
using a amylose resin affinity column, the fusion protein is cleaved with thrombin to remove the MBP tag, the cleavage mixture is applied to a Q anion-exchange column
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(lambdaDE3) cells
expression of GST-Endo IV fusion protein in Escherichia coli: wild-type and mutants W88R and S176N
-
gene sco4631, expression in Streptomyces lividans results in the loss of the genomic island that contains the dndA-E gene cluster, expression of N-terminally His6-tagged Sco4631 in Escherichia coli strain BL21(DE3)
-
into the pMal-c2x vector for expression in Escherichia coli TB1 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E118A
-
site-directed mutagenesis
S176N
-
mutant enzyme retains cleavage activity (17.5% of that of wild-type Endo IV), but loses the polarized and restricted cleavage of a dCs tract. Escherichia coli cells expressing the intact Endo IV mutant enzyme are viable and, in contrast to wild-type Endo IV, the mutant enzyme does not show detrimental effect on the host cells
W88R
-
mutant enzyme shows no enzymatic activity (less than 0.4% of that of wild-type Endo IV). Escherichia coli cells expressing the intact Endo IV mutant enzyme are viable and, in contrast to wild-type Endo IV, these mutant enzymes do not show detrimental effect on the host cells
G149D
-
site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities
H69A
-
site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation
G149D
-
site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities
-
H69A
-
site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation
-
additional information
-
analysis of enzyme activity in naturally occurring mutants of Escherichia coli compared to wild-type strain enzymes, overview
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