Information on EC 3.1.21.2 - deoxyribonuclease IV

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY
3.1.21.2
-
RECOMMENDED NAME
GeneOntology No.
deoxyribonuclease IV
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Endonucleolytic cleavage of ssDNA at apurinic/apyrimidinic sites to 5'-phosphooligonucleotide end-products
show the reaction diagram
-
-
-
-
Endonucleolytic cleavage of ssDNA at apurinic/apyrimidinic sites to 5'-phosphooligonucleotide end-products
show the reaction diagram
reaction mechanism
-
Endonucleolytic cleavage of ssDNA at apurinic/apyrimidinic sites to 5'-phosphooligonucleotide end-products
show the reaction diagram
reaction mechanism modelling
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of phosphoric ester
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
deoxriboendonuclease
-
-
-
-
DNA-adenine-transferase
-
-
-
-
E. coli endonuclease IV
-
-
-
-
endodeoxyribonuclease IV
-
-
-
-
nuclease, endodeoxyribo-oder redoxyendonuclease
-
-
-
-
T4 endonuclease IV
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37211-67-9
-
58591-37-0
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Escherichia coli phage, i.e. Coliphage T4
-
-
Manually annotated by BRENDA team
B.; phage T4 infected
-
-
Manually annotated by BRENDA team
gene nfo
-
-
Manually annotated by BRENDA team
gene nfo; wild type and nfo mutant
-
-
Manually annotated by BRENDA team
several strains
-
-
Manually annotated by BRENDA team
Escherichia coli AB1157
gene nfo
-
-
Manually annotated by BRENDA team
Escherichia coli B.
B.
-
-
Manually annotated by BRENDA team
gene sco4631
-
-
Manually annotated by BRENDA team
Streptomyces coelicolor A(3)2
gene sco4631
-
-
Manually annotated by BRENDA team
strain MSB8, thermophilic bacterium
Uniprot
Manually annotated by BRENDA team
Thermotoga maritima DSM 3109
-
Uniprot
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
-
phenotype with reduced DNA repair activity of enzyme-deficient mutant strain BW527, overview
physiological function
-
endonuclease IV recognizes and cleaves DNA on the 5' side of various oxidatively damaged bases via nucleotide incision repair (NIR), endonuclease IV is the main base excision repair enzyme involved in DNA damage induced by UVA radiation and stannous chloride
physiological function
-
in the nucleotide incision repair pathway, an apurinic/apyrimidinic endonuclease incises duplex DNA 5' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV is involved in both base excision repair and nucleotide incision repair pathways, overview
physiological function
-
in vitro endonuclease activity of the Escherichia coli McrA homologue that specifically cleaves S-modified DNA
physiological function
P9WQ13
the enzyme plays a crucial role in defense against oxidative stress
physiological function
Streptomyces coelicolor A(3)2
-
in vitro endonuclease activity of the Escherichia coli McrA homologue that specifically cleaves S-modified DNA
-
physiological function
-
the enzyme plays a crucial role in defense against oxidative stress
-
physiological function
Escherichia coli AB1157
-
endonuclease IV recognizes and cleaves DNA on the 5' side of various oxidatively damaged bases via nucleotide incision repair (NIR), endonuclease IV is the main base excision repair enzyme involved in DNA damage induced by UVA radiation and stannous chloride, in the nucleotide incision repair pathway, an apurinic/apyrimidinic endonuclease incises duplex DNA 5' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV is involved in both base excision repair and nucleotide incision repair pathways, overview
-
malfunction
Escherichia coli AB1157
-
phenotype with reduced DNA repair activity of enzyme-deficient mutant strain BW527, overview
-
additional information
-
a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks
additional information
-
Nfo catalyzed nucleotide incision and 3'-5' exonuclease activities are genetically coupled. modeling of nucleotide incision coupled to 3'-5' exonuclease activity preventing formation of lethal double-strand breaks when repairing bi-stranded clustered DNA damage, overview
additional information
Escherichia coli AB1157
-
Nfo catalyzed nucleotide incision and 3'-5' exonuclease activities are genetically coupled. modeling of nucleotide incision coupled to 3'-5' exonuclease activity preventing formation of lethal double-strand breaks when repairing bi-stranded clustered DNA damage, overview
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
21-mer C-Grec + H2O
?
show the reaction diagram
Escherichia coli, Escherichia coli AB1157
-
-
-
-
?
5'-end-labeled 19 nucleotide single-stranded DNA + H2O
?
show the reaction diagram
P9WQ13
-
-
-
?
5'-end-labeled 29 nucleotide double-stranded DNA + H2O
?
show the reaction diagram
P9WQ13
92% cleavage after 30 min
-
-
?
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
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
in vivo the enzyme produces mostly double-stranded fragments with a few internal nicks
-
-
ir
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides, hydrolysis of a phosphodiester bond near an alkylated base in native DNA without single breaks in the region, the enzyme makes predominantly single-strand break, and a few double-strand breaks, the ratio is 3.7:1
-
-
ir
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
the enzyme cleaves sites of DNA with conserved sequence elements to both the left and the right of the cleaved bonds comprising 16 basepairs with some variability tolerated, nicking of a single and both strands, the latter in two nicking events, substrate specificity, overview, in vitro the enzyme produces mostly single-stranded nicks
-
-
ir
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
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
preference for single-stranded DNA, denatured lambda-DNA, breaks exlusively adjacent to cytosine residues, no activity on denatured T4 DNA
about 150 nucleotides long
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
breakdown of host cell DNA occuring after T4 phage infection
-
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
the Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli
-
-
?
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
Endo IV catalyzes specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency dependent on the surrounding nucleotide sequence. The enzyme preferentially targets 5'-dTdCdA-3' but tolerates various combinations of individual nucleotides flanking this trinucleotide sequence. Endo IV preferentially recognizes short nucleotide sequences containing 5'-TdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA
-
-
?
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonuclease IV cleaves single-stranded (ss) DNA in a dC-specific manner. The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli
-
-
?
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
the enzyme cleaves single-stranded (ss) DNA in a dC-specific manner. Endo IV cleaves ssDNAs exclusively at the 5-proximal dC where a sequence comprises dC residues both at the 5 proximal and 3 proximal positions (a dCs tract-dependent cleavage). The dCs tract-dependent cleavage is efficient and occurs when a dCs tract has at least 6 bases. Some dCs tracts larger than 6 bases behave as that of 6 bases (an extended dCs tract), while some others do not. One decameric dCs tract is shown to be cleavable in a dCs tract-dependent manner, but that with 13 dCs is not. The dCs tract-dependent cleavage is enhanced by the presence of a third dC residue at least for a 6 or 7 dCs tract. In contrast to the dCs tract-dependent cleavage, a dCs tract-independent one is generally inefficient and if two modes are possible for a substrate DNA, a dCs tract-dependent mode prevails
-
-
?
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
Escherichia coli B.
-
preference for single-stranded DNA, denatured lambda-DNA, breaks exlusively adjacent to cytosine residues, no activity on denatured T4 DNA
about 150 nucleotides long
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
Escherichia coli B.
-
breakdown of host cell DNA occuring after T4 phage infection
-
ir
double-stranded nativeDNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
additional information
?
-
-
DNA substrate specificity, overview
-
-
-
additional information
?
-
-
endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli
-
-
-
additional information
?
-
P0A6C1
role for Endo IV to counteract DNA damage induced by the oxidative counterpart of UVB in Escherichia coli
-
-
-
additional information
?
-
-
reaction proceeds through a synchronous bimolecular (ANDN) mechanism with reaction free energy and barrier of -3.5 and 20.6 kcal/mol.In the course of the reaction the trinuclear active site of endonuclease IV undergoes dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands
-
-
-
additional information
?
-
-
a His-tagged derivative of ScoA3McrA cleaves S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Sco4631 cleaves DNA at multiple possible sites on either side of the S-modification. Double-strand cleavage occurs 16-28 nucleotides away from the phosphorothioate links
-
-
-
additional information
?
-
-
EndoII primarily catalyzes single-stranded nicking of DNA, 5 to 10fold less frequently double-stranded breaks are produced
-
-
-
additional information
?
-
-
Nfo-catalyzed nucleotide incision repair and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue
-
-
-
additional information
?
-
P9WQ13
endonuclease activity of the enzyme is about 2fold higher for double-stranded DNA in comparison to single-stranded DNA
-
-
-
additional information
?
-
P9WQ13
the enzyme also shows 3'->5' exonuclease activity
-
-
-
additional information
?
-
-
the enzyme hydrolyzes the 3'-nucleotide in the following order: nick > 3'-blunt > 3'-recessed > single stranded DNA
-
-
-
additional information
?
-
Streptomyces coelicolor A(3)2
-
a His-tagged derivative of ScoA3McrA cleaves S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Sco4631 cleaves DNA at multiple possible sites on either side of the S-modification. Double-strand cleavage occurs 16-28 nucleotides away from the phosphorothioate links
-
-
-
additional information
?
-
P9WQ13
endonuclease activity of the enzyme is about 2fold higher for double-stranded DNA in comparison to single-stranded DNA, the enzyme also shows 3'->5' exonuclease activity
-
-
-
additional information
?
-
Escherichia coli AB1157
-
Nfo-catalyzed nucleotide incision repair and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
double-stranded alkylated DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
in vivo the enzyme produces mostly double-stranded fragments with a few internal nicks
-
-
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
breakdown of host cell DNA occuring after T4 phage infection
-
ir
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
the Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli
-
-
?
single-stranded DNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
Escherichia coli B.
-
breakdown of host cell DNA occuring after T4 phage infection
-
ir
double-stranded nativeDNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
endonucleolytic cleavage of DNA into 5'-phosphooligonucleotides
-
-
ir
additional information
?
-
-
endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli
-
-
-
additional information
?
-
P0A6C1
role for Endo IV to counteract DNA damage induced by the oxidative counterpart of UVB in Escherichia coli
-
-
-
additional information
?
-
-
a His-tagged derivative of ScoA3McrA cleaves S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Sco4631 cleaves DNA at multiple possible sites on either side of the S-modification. Double-strand cleavage occurs 16-28 nucleotides away from the phosphorothioate links
-
-
-
additional information
?
-
-
EndoII primarily catalyzes single-stranded nicking of DNA, 5 to 10fold less frequently double-stranded breaks are produced
-
-
-
additional information
?
-
-
Nfo-catalyzed nucleotide incision repair and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue
-
-
-
additional information
?
-
Streptomyces coelicolor A(3)2
-
a His-tagged derivative of ScoA3McrA cleaves S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Sco4631 cleaves DNA at multiple possible sites on either side of the S-modification. Double-strand cleavage occurs 16-28 nucleotides away from the phosphorothioate links
-
-
-
additional information
?
-
Escherichia coli AB1157
-
Nfo-catalyzed nucleotide incision repair and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
P9WQ13
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
Mg2+
-
stimulates
Mg2+
-
absolutely required for nicking activity
Mg2+
Q9WYJ7
contains Mg2+
Mg2+
P9WQ13
maximally stimulated in the presence of Mg2+
MgCl2
-
absolute requirement, no activity in absence, optimal concentration: 0.01 M
Mn2+
-
0.01 M can replace Mg2+
Mn2+
-
stimulates
Mn2+
-
required for activity
Mn2+
Q9WYJ7
contains Mn2+, the enzyme with Mn2+ and Zn2+ is more active than with only Zn2+
Zn2+
-
dependent on
Zn2+
Q9WYJ7
contains Zn2+
Mn2+
P9WQ13
slight stimulation
additional information
-
the enzyme shows no absolute requirement for divalent cations, no effect by Ca2+
additional information
P9WQ13
not stimulated by Zn2+, Co2+; the enzyme shows metal ion independent 3'->5' exonuclease activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(NH4)2SO4
-
0.05 M
4-Chloromercuriphenylsulfonate
-
-
8-hydroxyquinoline
-
inhibits cleavage of alkylated dsDNA
diethyldithiocarbamic acid
-
-
dithiothreitol
P9WQ13
complete inhibition at 50 mM
EDTA
-
inhibits cleavage of non-alkylated dsDNA, 70% inhibition at 0.1 mM
EDTA
Q72KH8
in the presence of 10 mM EDTA enzymatic activity is completely eliminated
EDTA
P9WQ13
complete inhibition at 50 mM
KCl
-
0.05 M
NaCl
-
0.05 M
NH4Cl
-
0.05 M
p-hydroxymercuribenzoate
-
-
potassium phosphate buffer
-
pH 8.3
KCl
-
24% inhibition at 0.1 M, 42% at 0.2 M, and 64% at 0.3 M
additional information
-
enzyme inhibition by chelating and sulfhydryl agents, but not by tRNA and caffeine, DTT or 2-mercaptoethanol protect against inhibition by sulfhydryl reagents
-
additional information
P9WQ13
the enzyme is tolerant to EDTA up to 2 mM
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
IMAGE
0.000025
21-mer C Grec
-
wild-type enzyme, pH 7.6, 37C
-
0.0000272
21-mer C Grec
-
mutant H69A, pH 7.6, 37C, in presence of Zn2+
-
0.000028
21-mer C Grec
-
mutant G149D, pH 7.6, 37C
-
0.00000691
5'-end-labeled 29 nucleotide double-stranded DNA
P9WQ13
at pH 8.0 and 37C
-
additional information
additional information
-
kinetics
-
additional information
additional information
-
effects of dC-flanking sequences of the substrate on kinetic parameters of Endo IV
-
additional information
additional information
-
Km-values of Endo IV with with oligonucleotide substrates containing a target sequence and with various dC1-dC6 tracts and the effects of third dC residue in the tract
-
additional information
additional information
-
Km-values of the wild-type and S176N mutant forms of Endo IV are determined with series of 25-base oligonucleotides
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0003
21-mer C Grec
-
mutant H69A, pH 7.6, 37C, in presence of Zn2+
-
0.0088
21-mer C Grec
-
mutant G149D, pH 7.6, 37C
-
0.28
21-mer C Grec
-
wild-type enzyme, pH 7.6, 37C
-
0.0193
5'-end-labeled 29 nucleotide double-stranded DNA
P9WQ13
at pH 8.0 and 37C
-
additional information
additional information
-
effects of dC-flanking sequences of the substrate on kinetic parameters of Endo IV
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.012
21-mer C Grec
-
mutant H69A, pH 7.6, 37C, in presence of Zn2+
0
0.317
21-mer C Grec
-
mutant G149D, pH 7.6, 37C
0
8
21-mer C Grec
-
wild-type enzyme, pH 7.6, 37C
0
2790
5'-end-labeled 29 nucleotide double-stranded DNA
P9WQ13
at pH 8.0 and 37C
0
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
relative activity in several Escherichia coli wild-type and mutant strains, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
Q72KH8
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
LITERATURE
38
-
assay at
PDB
SCOP
CATH
ORGANISM
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
LITERATURE
29000
Q72KH8
determined by SDS-PAGE
683117
32400
Q9WYJ7
SDS-PAGE
706967
additional information
-
sucrose density gradient sedimentation equilibrium analysis
665452
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
P9WQ13
x * 26800, SDS-PAGE
tetramer
-
4 * 16000
?
-
x * 26800, SDS-PAGE
-
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
Q9WYJ7
using the sitting-drop vapor-diffusion method
Q9WYJ7
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
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
665452
45
-
10 min, fraction IV, loss of 10% activity
665452
60
-
10 min, fraction IV, complete inactivation
665452
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
Q72KH8
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
P9WQ13
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
Q72KH8
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of GST-Endo IV fusion protein in Escherichia coli: wild-type and mutants W88R and S176N
-
expressed in Escherichia coli BL21(lambdaDE3) cells
P9WQ13
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)
-
expressed in Escherichia coli
-
into the pMal-c2x vector for expression in Escherichia coli TB1 cells
Q72KH8
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
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
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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
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site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities
G149D
Escherichia coli AB1157
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site-directed mutagenesis, the mutant is deficient in both nucleotide incision repair and exonuclease activities
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H69A
Escherichia coli AB1157
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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
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H508A
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catalytically inactive mutant
H508A
Streptomyces coelicolor A(3)2
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catalytically inactive mutant
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H69A
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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
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analysis of enzyme activity in naturally occurring mutants of Escherichia coli compared to wild-type strain enzymes, overview