Information on EC 3.2.2.20 - DNA-3-methyladenine glycosylase I

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

EC NUMBER
COMMENTARY hide
3.2.2.20
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RECOMMENDED NAME
GeneOntology No.
DNA-3-methyladenine glycosylase I
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of alkylated DNA, releasing 3-methyladenine
show the reaction diagram
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-
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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
89287-37-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
strain 168
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Manually annotated by BRENDA team
strain 168
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Manually annotated by BRENDA team
strain BW 9062
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Manually annotated by BRENDA team
serovar lai strain 56601
UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
mouse
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Manually annotated by BRENDA team
no activity in Escherichia coli
Saccharomyces pombe
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5'-CGATAGCATCCT[hypoxanthine]CCTTCTCTCCAT-3' + H2O
?
show the reaction diagram
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-
-
-
?
alkylated DNA + H2O
1,N6-ethenoadenine + ?
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + 3-methylguanine + ?
show the reaction diagram
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AlkC is specific for 3-methylpurines
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?
alkylated DNA + H2O
3-methyladenine + ?
show the reaction diagram
alkylated DNA + H2O
3-methylguanine + ?
show the reaction diagram
-
-
-
?
alkylated DNA + H2O
7-methylguanine + ?
show the reaction diagram
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
1,N6-ethenoadenine + ?
show the reaction diagram
alkylated DNA + H2O
3-methyladenine + ?
show the reaction diagram
alkylated DNA + H2O
3-methylguanine + ?
show the reaction diagram
Q8EZM1
-
-
-
?
alkylated DNA + H2O
7-methylguanine + ?
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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CaCl2 stimulates enzyme activity
Mn2+
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MnCl2 stimulates enzyme activity
Zn2+
binds Zn2+ extremely tight to stabilize the HhH domain
additional information
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does not require divalent metal ions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11-benzyl-11H-pyrido[3'2':5-6]pyrazino[2-3-b]indole
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3-Methyladenine
apurinic-DNA
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benzyl-3H-imidazo[4,5-e][1,2,4]triazolo[1,5c][1,2,3]triazin-8(7H)-one
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Double-stranded DNA
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N-(4-tert-butyl-2-pyridinyl)-1-((4-tert-butyl-2-pyridinyl)imino)-1H-isoindol-3-amine
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N-(5-methyl-2-pyridinyl)-1-((5-methyl-2-pyridinyl)imino)-1H-isoindol-3-amine
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N-benzhydryl-9H-purin-6-amine
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N-ethylmaleimide
N2,N2-dimethyl-6,7-diphenyl-2,4-pteridinediamine
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N4,N7,1-tribenzyl-1H-imidazo[4,5-d]pyridazine-4,7-diamine
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N6-(5H-indeno[1,2-b]pyridin-3-ylmethyl)pteridine-2,4,6-triamine
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N6-benzhydryl-9H-purine-2-6-diamine
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N7-benzhydryl-3H-imidazo[4,5-b]pyridine-5,7-diamine
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p-hydroxymercuribenzoate
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p-mercuribenzoate
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spermidine
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additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-Methylguanine
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adenine
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Caffeine
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upstream region of the mbaag gene
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transcription factor BCG0878c stimulates the base excision activity of the enzyme and stimulates its binding activity for damaged DNA
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000006 - 0.000014
3-methyladenine residues in alkylated DNA
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Homo sapiens
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single-turnover excision kinetics of wild-type and mutant enzymes, overview
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6
3-Methyladenine
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pH 7.8, 37°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0004
3-benzyladenine
Leptospira interrogans
Q8EZM1
pH and temperature not specified in the publication
0.0023
3-butyladenine
Leptospira interrogans
Q8EZM1
pH and temperature not specified in the publication
0.05
3-ethyladenine
Leptospira interrogans
Q8EZM1
pH and temperature not specified in the publication
1.5
3-Methyladenine
Leptospira interrogans
Q8EZM1
pH and temperature not specified in the publication
0.005
3-propyladenine
Leptospira interrogans
Q8EZM1
pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000242
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
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7 - 9
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more active in 0.07 M HEPES-KOH buffer than in 0.07 M Tris-HCl buffer
7.2 - 7.8
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similar activity in either HEPES-KOH or Tris-HCl buffers
7.8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
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no activity below pH 5.0 and above pH 10.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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isoelectric focusing
6.5
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predicted from amino acid composition
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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gel filtration
22000
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gel filtration, Svedberg equation
22500
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SDS-PAGE
22703
x * 22703, calculated from amino acid sequence
28000
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x * 28000, AlkC, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 22500, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in unliganded form and in a ternary product complex with abasic DNA and 3-methyladenine. The abasic DNA is not flipped into the enzyme's active site, instead conformational relaxation must occur in the DNA upon base hydrolysis
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wild type and mutant enzyme Y16F in complex with 3-methyladenine, sitting drop vapor diffusion method, using 0.1 M Tris-HCl pH 8.5, 1.8 M ammonium sulfate, 0.2 M Li2SO4
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48
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50% inactivation requires 5 min
60
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heat labile, during a 2 min incubation reduces activity to less than 2%
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
repeated freezing and thawing does not effect any apparent loss in enzyme activity
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very unstable
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, stored frozen in buffer containing 1 mg/ml bovine serum albumin, stable for more than 6 months
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-20°C, stored in 50% glycerol, undergoes complete inactivation in 3-4 weeks
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-70°C, buffer containing 20% glycerol, stable for long term storage
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-70°C, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography
overproducing strain, carrying the tag gene on a multicopy plasmid
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partially
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recombinant protein, expressed in Escherichia coli
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Aag cDNA cloned and expressed in Escherichia coli
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aag gene cloned in pUC19and expressed in Escherichia coli
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cloned and bacterially expressed as GST-MPG fusion protein
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cloned and overexpressed in Salmonella typhimurium
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complete nucleotide sequence of the tag gene determined
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expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expressed in in a glycosylase-deficient Escherichia coli strain MV1932
expression in Escherichia coli BL2, AlkC
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genetic mapping of the tag gene
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integration and expression of the tag gene with retroviral vectors into chinese hamster V79 cells by liposome mediated transfection and into murine haemopoeitic stemm cells by cocultivation with a lipofected, virus-packaging cell line, stable transfectants are more resistant to cytotoxic effects of methylmethanesulfonate and N-methyl-nitrosourea than their parent cell lines
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tag gene cloned and expressed in murine fibroblasts NIH-3T3
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TAG gene sequence PCR amplified and cloned into pET21a
tag+ gene cloned and sequenced
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wild-type TAG overexpressed from pET21a(TAG) in Escherichia coli BL21(DE3)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transcription factor BCG0878c negatively regulates the expression of the enzyme (4fold)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D18A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
E38A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
H17A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
W21A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
W46A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
Y13A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
Y16A
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TAG mutant, prepared with QuikChange double-stranded mutagenesis kit
D170N
Saccharomyces pombe
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the mutation substantially decreases enzymatic activity without abolishing it
I147V/T151A/A206S
Saccharomyces pombe
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the mutant retains base excision activity toward alkylated DNA substrates, but its level of activity is decreased compared to the wild type
K126N/E130G/N175D
Saccharomyces pombe
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the mutant retains base excision activity toward alkylated DNA substrates, but its level of activity is decreased compared to the wild type
L122M
Saccharomyces pombe
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the mutant retains base excision activity toward alkylated DNA substrates, but its level of activity is decreased compared to the wild type
N42Y/V165A
Saccharomyces pombe
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the mutant retains base excision activity toward alkylated DNA substrates, but its level of activity is decreased compared to the wild type
R149K
Saccharomyces pombe
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the mutant retains base excision activity toward alkylated DNA substrates, but its level of activity is decreased compared to the wild type
additional information
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construction of a truncated DELTA80AAG enzyme mutant, substrate specificity in comparison to the wild-type enzyme shows that both the full-length and truncated AAG excise 1,N2-ethenoguanine, albeit weakly, from duplex DNA, while uracil is excised from both single- and double-stranded DNA, but only by full-length AAG, single-turnover excision kinetics, overview
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