Information on EC 3.2.2.21 - DNA-3-methyladenine glycosylase II

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The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria

EC NUMBER
COMMENTARY hide
3.2.2.21
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RECOMMENDED NAME
GeneOntology No.
DNA-3-methyladenine glycosylase II
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of alkylated DNA, releasing 3-methyladenine, 3-methylguanine, 7-methylguanine and 7-methyladenine
show the reaction diagram
involved in the removal of alkylated bases from DNA in Escherichia coli (cf. EC 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase)
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of N-glycosyl bond
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SYSTEMATIC NAME
IUBMB Comments
alkylated-DNA glycohydrolase (releasing methyladenine and methylguanine)
Involved in the removal of alkylated bases from DNA in Escherichia coli (cf. EC 2.1.1.63 methylated-DNA---[protein]-cysteine S-methyltransferase).
CAS REGISTRY NUMBER
COMMENTARY hide
89287-38-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
AB1157
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-
Manually annotated by BRENDA team
strain BW 9062
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Manually annotated by BRENDA team
MV1161
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
no activity in Escherichia coli
alk mutants
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-
Manually annotated by BRENDA team
yeast
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-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2-propanediol-DNA + H2O
?
show the reaction diagram
-
-
-
-
?
1,3-propanediol-DNA + H2O
?
show the reaction diagram
-
-
-
-
?
alkylated DNA + H2O
3-methyl-2'-deoxyadenosine + 3-deaza-3-methyl-2'-deoxyadenosine + DNA
show the reaction diagram
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the sequences of the oligos used in the assay are 5'-CGATAGCATCCTYCCTTCTCTCCAT-3', where Y is the location of the lesion base, and 5'-ATGGAGAGAAGGZAGGATGCTATCG-3' for the complementary strand, where Z is the base opposite the lesion
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-
?
alkylated DNA + H2O
3-methyladenine + 3-methylguanine + 7-methyladenine + 7-methylguanine + DNA
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + 3-methylguanine + 7-methylguanine + 7-methyladenine + ?
show the reaction diagram
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AlkA has significant glycosylase activity towards each of the normal bases in DNA. AlkA binds nonspecifically to DNA and neither mismatches nor 7-methylguanine lesions are specifically recognized in the ground state
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?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + 3-methylguanine
show the reaction diagram
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AlkD has high activity towards 7-methylguanine but removes 3-methylguanine more slowly as compared with Escherichia coli AlkA
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-
?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + 7-methyladenine + DNA
show the reaction diagram
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-
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?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + ?
show the reaction diagram
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ratio of 3-methyladenine/7-methylguanine is 29:1 for wild-type enzyme, 24:1 for mutant enzyme N169S and 26:1 for mutant enzyme N169A, no production of 7-methylguanine is detected with mutant enzyme N169D
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?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + DNA
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + hypoxanthine + DNA
show the reaction diagram
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-
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?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + O2-methylthymine + O2-methylcytosine + 1,N6-ethenoadenine + hypoxanthine + DNA
show the reaction diagram
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-
-
-
?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + O2-methylthymine + O2-methylcytosine + DNA
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + ?
show the reaction diagram
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-
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?
alkylated DNA + H2O
3-methyladenine + N3-methylcytosine + 1,N6-ethenoadenine + DNA
show the reaction diagram
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excision of N3-methylcytosine is about twice as fast as excision of 3-methyladenine, and the rate for 1,N6-ethenoadenine is further 5-10 times higher
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?
alkylated DNA + H2O
7-methyladenine + 7-methylguanine + 3-methyladenine + 3-methylguanine + purine + 6-chloropurine + xanthine + DNA
show the reaction diagram
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7-methylguanine is cleaved the most quickly
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?
alkylated DNA + H2O
?
show the reaction diagram
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the efficiency of 3-methyladenine and 3-methylguanine removal was 510 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-methylguanine removal is similar to the two enzymes
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?
DNA treated with beta-[3H]propiolactone + H2O
N1-(carboxyethyl)adenine + N7-(carboxymethyl)guanine
show the reaction diagram
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-
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?
duplex oligonucleotide substrate containing ethenoadenine + H2O
ethenoadenine + oligonucleotide
show the reaction diagram
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-
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?
duplex oligonucleotide substrate containing ethenoadenine and hypoxanthine + H2O
ethenoadenine + hypoxanthine + oligonucleotide
show the reaction diagram
ethanol-DNA + H2O
?
show the reaction diagram
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best substrate
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-
?
ethylene glycol-DNA + H2O
?
show the reaction diagram
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-
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?
glycerol-DNA + H2O
?
show the reaction diagram
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-
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?
methanol-DNA + H2O
?
show the reaction diagram
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?
propanol-DNA + H2O
?
show the reaction diagram
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?
synthetic oligosaccharide + H2O
?
show the reaction diagram
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synthetic oligosaccharide containing a single ethano or etheno adduct (3,N4-ethanocytosine, 1,N6-ethanoadenine, 3,N4-ethenocytosine or 1,N6-ethenoadenine), 20fold lower excision activity towards 3,N4-ethanocytosine and 1,N6-ethanoadenine than that towards their structurally analogous 3,N4-ethenocytosine or 1,N6-ethenoadenine. The enzyme is capable of excising the ethano base paired with any of the four natural bases
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?
additional information
?
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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
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + DNA
show the reaction diagram
Q9RRB0
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?
alkylated DNA + H2O
3-methyladenine + 7-methylguanine + O2-methylthymine + O2-methylcytosine + DNA
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + ?
show the reaction diagram
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-
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?
alkylated DNA + H2O
7-methyladenine + 7-methylguanine + 3-methyladenine + 3-methylguanine + purine + 6-chloropurine + xanthine + DNA
show the reaction diagram
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7-methylguanine is cleaved the most quickly
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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no absolute cofactor requirement
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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2 mM CaCl2, enzyme activity 103%
Cd2+
inhibitory above 0.05 mM. Reduced catalytic activity in presence of Zn2+ is not due to altered binding of substrate
K+
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optimal activity at pH 7.5 and 100 mM KCl
Mn2+
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2 mM MnCl2, enzyme activity 102%
Ni2+
inhibitory above 0.05 mM, but to lesser extent than Cd2+ or Zn2+. Reduced catalytic activity in presence of Zn2+ is not due to altered binding of substrate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxy-2'-fluoro-7-methylguanosine
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abasic-containing DNA
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potent inhibitor
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apurinic-DNA
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DNA containing 1-azaribose
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Double-stranded DNA
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Mg2+
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significant inhibition depending on its concentration, but independent of substrate type. Mg2+ at high but physiologic concentrations decreases the amount of active enzyme. Inhibition affects the Km value, but not Vmax, and is due to inhibition in substrate binding and can be reversed by EDTA. At low concentration, Mg2+ stimulates enzyme activity with substrate hypoxanthine, but not with ethenoadenine
N-ethylmaleimide
Ni2+
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0.001 mM
p-hydroxymercuribenzoate
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p-mercuribenzoate
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Pyrrolidine-containing oligonucleotide
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ZnSO4
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2 mM, enzyme activity 23%
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-Methyladenine
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5 mM, enzyme activity 110%
3-Methylguanine
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5 mM, enzyme activity 120%
N-methyl-N'-nitro-N-nitrosoguanidine
spermidine
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stimulates activity
spermine
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stimulates activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000024 - 0.0013
1,N6-ethenoadenine residues in alkylated DNA
0.0000092
3-methyladenine residues in alkylated DNA
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pH 8.0, 37C, release of 3-methyladenine
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0.000011
7-methylguanine residues in alkylated DNA
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pH 8.0, 37C, release of 7-methylguanine
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0.00000014 - 0.00003
alkylated DNA
0.000031 - 0.000053
alkylated DNA, treated with N-methyl-N'-nitro-N-nitrosoguanidine
0.0000354 - 0.042
duplex oligonucleotide substrate containing ethenoadenine and hypoxanthine
0.0000053
hypoxanthine
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at 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000833 - 0.0167
1,N6-ethenoadenine residues in alkylated DNA
0.00125
duplex oligonucleotide substrate containing ethenoadenine and hypoxanthine
Escherichia coli
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at 37C, in 50 mM Na-MES buffer (pH 6.1)
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0.0033
hypoxanthine
Homo sapiens
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at 37C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
30
duplex oligonucleotide substrate containing ethenoadenine and hypoxanthine
Escherichia coli
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at 37C, in 50 mM Na-MES buffer (pH 6.1)
14229
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00000008
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mutant Asn-152, expressed in Escherichia coli
0.000005
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mutant Asn-252, expressed in Escherichia coli
0.000007
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mutant Asn-255 and Asn-231, expressed in Escherichia coli
0.000009
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mutant Asn-259, expressed in Escherichia coli
0.00001
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mutant Asn-260, expressed in Escherichia coli
0.000013
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mutant Asn-112, expressed in Escherichia coli
0.000014
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wild-type Ndelta100Cdelta18, expressed in Escherichia coli
0.000018
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mutant Asn-310, expressed in Escherichia coli
0.000375
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-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3 - 7.4
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isoelectric focusing
9.65
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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normal, malignant and immortalized breast cells
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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immortalized, non-transformed breast epithelial cell line
Manually annotated by BRENDA team
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thymic carcinoma
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22000
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gel filtration, Svedberg equation
27000
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gel filtration
31000
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SDS-PAGE
32000
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SDS-PAGE
33000
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SDS-PAGE
34250
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predicted from nucleotide sequence
40000
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recombinant protein, expressed in Escherichia coli, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 25000, AlkD, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 1.8 A resolution. Structure contains two sodium ions in octahedral coordination
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sitting drop vapour diffusion method, with 85 mM HEPES (pH 7.5), 15% (w/v) PEG 4000, and 17% (v/v) glycerol, at 21C
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isoform AlkA2, hanging drop vapor diffusion method, using 1 M LiCl2, 0.1 M MES pH 6.0, 10% (w/v) PEG 6000
2.5 A crystal structure colmplexed to DNA, crystals belong to space group P3(1)21, cell dimensions a = B = 82.4 A and c = 199.7 A
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AlkA in complex with undamaged DNA, sitting drop vapor diffusion method, using 25-29% (w/v) polyethylene glycol 3350, 100 mM bis-Tris, pH 6.0-6.6, 200 mM Li2S04, and 3% (w/v) 6-aminocaproic acid, at 25C
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crystal structure of AlkA solved with the multiple isomorphous replacement method, crystals grown with sitting-drop vapor-diffusion technique, space group P2(1), cell dimensions a = 58.61 A, b = 76.93 A, c = 62.27 A
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hanging drop vapor difusion crystallization, space group P2(1), unit-cell parameters are a = 58.17, b = 76.27, c = 61.69, alpha = gamma = 90, beta = 109.98
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sitting drop method of vapour diffusion, monoclinic space group C2, a = 58.6 A, b = 76.8 A, c = 62.2 A
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AAG complexed to DNA
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expression in Saccharomyces cerevisiae
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
activity reduced by 50%
60
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heat stable, retains 40% of its original activity after 2 min incubation
90
-
60% enzyme activity retained after incubation for 1 h
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
very unstable
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stored in 50% glycerol, undergoes complete inactivation in 3-4 weeks
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-70C, purified enzyme remains stable for more than 4 years when stored in 20% glycerol,
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Mono Q column chromatography
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Ni-NTA column chromatography and Source S cation exchange column chromatography
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Ni-NTA column chromatography, heparin affinity column chromatography, and gel filtration
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nickel-loaded HisTrap column chromatography
purified from an overproducing strain
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purified from overproducing strains DH1 and HB101, harboring plasmid pYN3070
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purified from strain JM105 harboring pAlk10 plasmid
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recombinant protein, expressed from baculovirus
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recombinant protein, expressed in Escherichia coli
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recombinant protein, purified from Escherichia coli
SP Sepharose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alkAg+ gene cloned and sequenced
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cloned and expressed in Escherichia coli BL21(DE3)
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construction of recombinant GST-MPG fusion proteins
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expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21(DE3) Codon Plus cells
expressed in Escherichia coli HMS174 cells
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expression by adenoviral system and targeting of enzyme to mitochondria or nucleus of primary astrocytes
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expression in Escherichia coli BL21, AlkD
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gene afalkA, AF2117 ORF cloned and overexpressed in Escherichia coli, gene function suppresses the alkylation sensitivity in Escherichia coli tag alkA double mutants
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MPG deletion mutant N-DELTA100C-DELTA18 overexpressed in Escherichia coli
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overexpressed in MDA-MB231 breast cancer cell line
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truncated MPG polypeptides expressed in Escherichia coli using vector pRSET, full length MPG protein cloned in eukaryotic baculovirus expression vector pVL1393 and host Sf9 insect cells used for expression
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D113N
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the mutation results in a 100fold decrease in the single-turnover rate of 7-methylguanine excision relative to the wild type enzyme
R148A
-
the mutation results in a 100fold decrease in the single-turnover rate of 7-methylguanine excision relative to the wild type enzyme
W218A
-
site-directed mutagenesis
N169A
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ratio of 3-methyladenine/7-methylguanine is 29:1 for wild-type enzyme and 26:1 for mutant enzyme N169A, 30fold lower activity than wild-type enzyme. Expression of the N169A in Saccharomyces cerevisiae during methyl methanesulfonate exposure results in greater sensitivity, greater mutation induction following exposure to methyl methanesulfonate and more strand breaks in vivo
N169D
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ratio of 3-methyladenine/7-methylguanine is 29:1 for wild-type enzyme, 100fold lower production of 3-methyladeine and no production of 7-methylguanine is detected with mutant enzyme N169D. When expressed in Saccharomyces cerevisiae, the N169D variant provides better protection against methyl methanesulfonate toxicity than wild-type enzyme. Fewer strand breaks in vivo are seen in the presence of the N169D variant following exposure to methyl methanesulfonate
N169S
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ratio of 3-methyladenine/7-methylguanine is 29:1 for wild-type enzyme and 24:1 for mutant enzyme N169S, 30fold lower activity than wild-type enzyme. Expression of the N169S in Saccharomyces cerevisiae during methyl methanesulfonate exposure results in greater sensitivity, greater mutation induction following exposure to methyl methanesulfonate and more strand breaks in vivo
D112N
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site-directed mutagenesis
D152N
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site-directed mutagenesis
D252N
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site-directed mutagenesis
D259N
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site-directed mutagenesis
D260N
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site-directed mutagenesis
D310N
-
site-directed mutagenesis
additional information
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recombinant expression of enzyme and targeting to mitochondria or nucleus of primary astrocytes. Increasing enzyme activity significantly increases base excision repair kinetics in both the mitochondria and nuclei. Increased nuclear enzyme activity results in cell death in astrocyte cultures treated with methylnitrososurea
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
inhibition by Cd2+, Ni2+, or Zn2+ can be reversed by EDTA
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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