Information on EC 2.1.1.63 - methylated-DNA-[protein]-cysteine S-methyltransferase

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

EC NUMBER
COMMENTARY hide
2.1.1.63
-
RECOMMENDED NAME
GeneOntology No.
methylated-DNA-[protein]-cysteine S-methyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
DNA (containing 6-O-methylguanine) + protein L-cysteine = DNA (without 6-O-methylguanine) + protein S-methyl-L-cysteine
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
SYSTEMATIC NAME
IUBMB Comments
DNA-6-O-methylguanine:[protein]-L-cysteine S-methyltransferase
This protein is involved in the repair of alkylated DNA. It acts only on the alkylated DNA (cf. EC 3.2.2.20, DNA-3-methyladenine glycosidase I and EC 3.2.2.21, DNA-3-methyladenine glycosidase II). This enzyme catalyses only one turnover and therefore is not strictly catalytic.
CAS REGISTRY NUMBER
COMMENTARY hide
77271-19-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
KOD1
-
-
Manually annotated by BRENDA team
KOD1
-
-
Manually annotated by BRENDA team
strain JD52
-
-
Manually annotated by BRENDA team
strain JD52
-
-
Manually annotated by BRENDA team
strain HB8
-
-
Manually annotated by BRENDA team
Jp 163 A
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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
DNA (containing 6-O-benzylguanine) + protein L-cysteine
DNA (without 6-O-benzylguanine) + protein S-benzyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA (containing 6-O-carboxymethylguanine) + protein L-cysteine
DNA (without 6-O-carboxymethylguanine) + protein S-carboxymethyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA (containing 6-O-methylguanine) + protein L-cysteine
DNA (without 6-O-methylguanine) + protein S-methyl-L-cysteine
show the reaction diagram
DNA (containing 6-O-methylguanine) + [protein] L-cysteine
DNA (without 6-O-methylguanine) + [protein] S-methyl-L-cysteine
show the reaction diagram
DNA (containing 6-O-methylguanine) + [protein]-L-cysteine
DNA (without 6-O-methylguanine) + [protein]-S-methyl-L-cysteine
show the reaction diagram
DNA (containing O6-chloroethylguanine) + protein L-cysteine
DNA (without O6-chloroethylguanine) + protein S-chloroethyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA (containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine) + protein L-cysteine
DNA (without O6-[4-oxo-4-(3-pyridyl)butyl]guanine) + protein S-4-oxo-4-(3-pyridyl)butyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA containing 4-O-methylthymine + [protein-L-cysteine
DNA lacking 4-O-methylthymine + [protein]-S-methyl-L-cysteine
show the reaction diagram
DNA containing 4-O-methylthymine + [protein]-L-cysteine
DNA lacking 4-O-methylguanine + [protein]-S-methyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA containing 6-O-benzylguanine + [protein]-L-cysteine
DNA lacking 6-O-benzylguanine + [protein]-S-methyl-L-cysteine
show the reaction diagram
-
AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is not rate-limiting in the case of 6-O-benzylguanine and releases the dealkylated DNA
-
-
?
DNA containing 6-O-ethylguanine + [protein]-L-cysteine
DNA lacking 6-O-ethylguanine +[protein]-S-ethyl-L-cysteine
show the reaction diagram
DNA containing 6-O-methylguanine + [protein]-L-cysteine
DNA lacking 6-O-methylguanine + [protein]-S-methyl-L-cysteine
show the reaction diagram
DNA containing O6-(4-oxo-4-(3-pyridyl)butyl)guanine
?
show the reaction diagram
-
-
-
-
?
SNAP-Vista Green + [protein]-L-cysteine
guanine + [protein]-S-Vista Green-L-cysteine
show the reaction diagram
[N-[2-(2-[2-[(3-[[(2-amino-9H-purin-6-yl)oxy]methyl]phenyl)methoxy]ethoxy]ethoxy)ethyl]-5-(3,5-dimethyl-1H-pyrrol-2-yl-kappaN)-5-(3,5-dimethyl-2H-pyrrol-2-ylidene-kappaN)pentanamidato](difluoro)boron + [protein]-L-cysteine
[protein]-S-methyl-L-cysteine + ?
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
DNA (containing 6-O-carboxymethylguanine) + protein L-cysteine
DNA (without 6-O-carboxymethylguanine) + protein S-carboxymethyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA (containing 6-O-methylguanine) + protein L-cysteine
DNA (without 6-O-methylguanine) + protein S-methyl-L-cysteine
show the reaction diagram
DNA (containing 6-O-methylguanine) + [protein] L-cysteine
DNA (without 6-O-methylguanine) + [protein] S-methyl-L-cysteine
show the reaction diagram
DNA (containing 6-O-methylguanine) + [protein]-L-cysteine
DNA (without 6-O-methylguanine) + [protein]-S-methyl-L-cysteine
show the reaction diagram
DNA (containing O6-chloroethylguanine) + protein L-cysteine
DNA (without O6-chloroethylguanine) + protein S-chloroethyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA (containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine) + protein L-cysteine
DNA (without O6-[4-oxo-4-(3-pyridyl)butyl]guanine) + protein S-4-oxo-4-(3-pyridyl)butyl-L-cysteine
show the reaction diagram
-
-
-
-
?
DNA containing 4-O-methylthymine + [protein-L-cysteine
DNA lacking 4-O-methylthymine + [protein]-S-methyl-L-cysteine
show the reaction diagram
DNA containing 6-O-methylguanine + [protein]-L-cysteine
DNA lacking 6-O-methylguanine + [protein]-S-methyl-L-cysteine
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
the enzyme requires no cofactor
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zinc
-
in bacterial expression systems, recombinant hAGT is produced in increasingly larger quantities when growth media are supplemented with up to 0.1 mM ZnCl2. Metal-enriched hAGT samples have a 5fold increase in repair rate constant over conventionally purified protein samples and a 60fold increase over metal-stripped hAGT. Zinc confers a mechanistic enhancement to repair activity that does not result from an increase in substrate binding affinity. Zinc also provides conformational stability to hAGT that may influence its regulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-([2-amino-6-[(4-bromothiophen-2-yl)methoxy]-9H-purin-9-yl]methoxy)ethanol
-
-
2-amino-O4-benzyl-6,7-dimethylpteridine
-
potent
2-amino-O4-benzyl-6-formylpteridine
-
potent
2-amino-O4-benzyl-6-hydroxymethylpteridine
-
potent
2-amino-O4-benzylpteridine
-
potent
2-amino-O4-benzylpteridine-6-carboxylic acid
-
potent
4-[(4-bromothiophen-2-yl)methoxy]-1H-pyrazolo[3,4-d]pyrimidin-6-amine
-
-
4-[bis(2-chloroethyl)amino]-L-phenylalanine
-
hyperthermia enhances the inhibitory effects of L-phenylalanine mustard on melanoma cell growth
6-(1-benzofuran-2-ylmethoxy)-9H-purin-2-amine
-
-
6-(4,5,6,7-tetrahydro-1-benzothiophen-2-ylmethoxy)-9H-purin-2-amine
-
-
6-(naphtho[1,2-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
-
-
6-(naphtho[2,1-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
-
-
6-(phenanthro[9,10-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
-
-
6-O-benzylguanine
6-[(1-methyl-4-nitro-1H-pyrrol-2-yl)methoxy]-9H-purin-2-amine
-
-
6-[(4-bromothiophen-2-yl)methoxy]-9-(2-deoxy-beta-D-erythro-pentofuranosyl)-9H-purin-2-amine
-
-
7-[(4-bromothiophen-2-yl)methoxy]-2,3-dihydro-1H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine
-
-
9-beta-D-arabinofuranosyl-6-[(4-bromothiophen-2-yl)methoxy]-9H-purin-2-amine
-
-
alkyltransferase-like protein
-
inhibits the transfer of methyl groups to MGMT, thus the action of MGMT on 6-O-methylguanine in DNA, inhibition is reversible by prolonged incubation in the presence of MGMT
-
Br(CH2)2Br
-
inactivates purified AGT and mutant R128A to approximately the same extent; inactivates purified AGT and mutant R128A to approximately the same extent, small reduction in the loss of activity in the absence of DNA, but no effect at all in the presence of DNA, inactivates mutant Y114A much less than wild-type, and DNA completely prevents this inactivation, mutants P140K and Y158H are less inactivated than wild-type AGT, specifically in the presence of DNA
Br(CH2)3Br
-
mutant P140K requires higher concentrations than wild-type AGT for inactivation
Br(CH2)5Br
-
mutant P140K requires higher concentrations than wild-type AGT for inactivation
BrCH2Br
-
wild-type AGT and mutant P140K show no difference in sensitivity to BrCH2Br
BrCH2OAc
-
reacts with the enzyme at its cysteine acceptor site, abolishing its DNA repair activity, the formation of AGT-Cys145S-CH2Br by BrCH2OAc
CH2Br2
-
reacts with the enzyme at its cysteine acceptor site, abolishing its DNA repair activity
DNA (containing 6-O-carboxymethylguanine)
-
-
-
DNA (containing 6-O-methylguanine)
-
-
double-stranded oligonucleotides
-
6-O-methylguanine, 6-O-(4-fluorobenzyl)-guanine, 6-O-(3-fluorobenzyl)-guanine, 6-O-(2-fluorobenzyl)-guanine, 6-O-benzylguanine, 6-O-benzylhypoxanthine. IC50: 1.4-3.0 nM
-
formaldehyde
-
decreases activity at levels up to 3fold higher than the maximally allowed workplace concentration, no decrease at the maximally allowed level
methyl bromide
methyl iodide
-
can directly alkylate the active site of the enzyme, the agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic 6-O-alkylguanine residue in DNA in vivo
methyl isocyanate
-
methyl isocyanate resulting from base-catalyzed activation of VNP40101M inhibits the enzyme, thereby enhancing the yield of the DNA G-C interstrand crosslink responsible for the antitumor activity of this agent
N9-cyclopentyl-O6-(4-bromothenyl)guanine
-
efficient inhibitor of wild-type AGT
Na2SO4
Na3 citrate
Ni2+
-
purified protein is not very sensitive to this metal but the loss of AGT could contribute to the well-known carcinogenicity of nickel
O(6)-benzylguanine
-
BG
O4-(4-bromothenylpterin)
-
-
O4-benzylfolic acid
-
30times more active than O6-benzylguanine against the wild-type alkyltransferase, inactivation of P140K mutant alkyltransferase. Inhibitor shows promise as an agent for possible tumor-selective alkyltransferase inactivation superior toO6-benzylguanine as a chemotherapy adjuvant
O6-(1,2-thiazol-4-ylmethyl)guanine
-
-
O6-(1,3-oxazol-5-yl)guanine
-
-
O6-(1,3-thiazol-5-yl)guanine
-
-
O6-(1-benzofuran-2-ylmethyl)guanine
-
-
O6-(2-benzo[b]thienylmethyl)guanine
-
-
O6-(3-pyridyl)guanine
-
-
O6-(4-(2-chloropyridyl))guanine
-
-
O6-(4-bromothenyl)-8-oxaguanine
-
-
O6-(4-bromothenyl)-8-thiaguanine
-
-
O6-(4-bromothenyl)guanine
O6-(4-pyridyl)guanine
-
-
O6-alkylating drugs
-
O6-alkylating drugs deplete MGMT activity indirectly via alkylation of DNA
-
O6-benzyl-2'-deoxyguanosine
-
-
O6-benzylguanine
O6-furfurylguanine
-
-
O6-methylguanine
-
-
O6-methylguanine oligonucleotide
O6-thenylguanine
-
original Patrin, became Patrin-1
O6-[(1-methyl-1H-imidazol-5-yl)methyl]guanine
-
-
temozolomide
VNP40101M
-
methyl isocyanate resulting from base-catalyzed activation of VNP40101M inhibits the enzyme, thereby enhancing the yield of the DNA G-C interstrand crosslink responsible for the antitumor activity of this agent
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,2,3,4-diepoxybutane
-
induces AGT-DNA cross-linking, takes place exclusively at the cysteine residues within the protein
activator of protein kinase C
-
increases the level of AGT mRNA and increases resistance to N,N'-bis(2-chloroethyl)-N-nitrosourea in HeLa cells
-
dexamethasone
-
-
glucocorticoid
inhibitor of protein phosphatase
-
increases the level of AGT mRNA and increases resistance to N,N'-bis(2-chloroethyl)-N-nitrosourea in HeLa cells
-
ionizing radiation
-
induces AGT
-
N,N'-bis(2-chloroethyl)-N-nitrosourea
-
BCNU
additional information
-
DNA damage, ionizing radiation
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00077
DNA (containing 6-O-methylguanine)
pH 6.5, 50C
0.01
SNAP-Vista Green
pH 6.5, 50C
additional information
additional information
-
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.4
SNAP-Vista Green
Sulfolobus solfataricus
Q97VW7
pH 6.5, 50C
additional information
additional information
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00033
2-([2-amino-6-[(4-bromothiophen-2-yl)methoxy]-9H-purin-9-yl]methoxy)ethanol
Homo sapiens
-
-
0.000006
4-[(4-bromothiophen-2-yl)methoxy]-1H-pyrazolo[3,4-d]pyrimidin-6-amine
Homo sapiens
-
-
0.00008
6-(1-benzofuran-2-ylmethoxy)-9H-purin-2-amine
Homo sapiens
-
-
0.185
6-(4,5,6,7-tetrahydro-1-benzothiophen-2-ylmethoxy)-9H-purin-2-amine
Homo sapiens
-
-
0.000022
6-(naphtho[1,2-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
Homo sapiens
-
-
0.00005
6-(naphtho[2,1-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
Homo sapiens
-
-
0.0011
6-(phenanthro[9,10-b]thiophen-2-ylmethoxy)-9H-purin-2-amine
Homo sapiens
-
-
0.00055
6-[(1-methyl-4-nitro-1H-pyrrol-2-yl)methoxy]-9H-purin-2-amine
Homo sapiens
-
-
0.000095
6-[(4-bromothiophen-2-yl)methoxy]-9-(2-deoxy-beta-D-erythro-pentofuranosyl)-9H-purin-2-amine
Homo sapiens
-
-
0.000045
7-[(4-bromothiophen-2-yl)methoxy]-2,3-dihydro-1H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine
Homo sapiens
-
-
0.000115
9-beta-D-arabinofuranosyl-6-[(4-bromothiophen-2-yl)methoxy]-9H-purin-2-amine
Homo sapiens
-
-
0.0000018
DNA (containing 6-O-carboxymethylguanine)
Homo sapiens
-
in 50 mM Tris-HCl pH 8.3, at 37C
-
0.00000093
DNA (containing 6-O-methylguanine)
Homo sapiens
-
in 50 mM Tris-HCl pH 8.3, at 37C
0.0000014 - 0.000003
double-stranded oligonucleotides
Homo sapiens
-
6-O-methylguanine, 6-O-(4-fluorobenzyl)-guanine, 6-O-(3-fluorobenzyl)-guanine, 6-O-(2-fluorobenzyl)-guanine, 6-O-benzylguanine, 6-O-benzylhypoxanthine. IC50: 1.4-3.0 nM
-
0.0005
N9-cyclopentyl-O6-(4-bromothenyl)guanine
Homo sapiens
-
-
0.000025
O4-(4-bromothenylpterin)
Homo sapiens
-
-
0.00007
O6-(1,2-thiazol-4-ylmethyl)guanine
Homo sapiens
-
-
0.00035
O6-(1,3-oxazol-5-yl)guanine
Homo sapiens
-
-
0.000033
O6-(1,3-thiazol-5-yl)guanine
Homo sapiens
-
-
0.000035
O6-(1-benzofuran-2-ylmethyl)guanine
Homo sapiens
-
-
0.00003
O6-(2-benzo[b]thienylmethyl)guanine
Homo sapiens
-
-
0.00043
O6-(3-pyridyl)guanine
Homo sapiens
-
-
0.00004
O6-(4-(2-chloropyridyl))guanine
Homo sapiens
-
-
0.00024
O6-(4-bromothenyl)-8-oxaguanine
Homo sapiens
-
-
0.0000028
O6-(4-bromothenyl)-8-thiaguanine
Homo sapiens
-
-
0.0000001 - 0.0000034
O6-(4-bromothenyl)guanine
0.00013
O6-(4-pyridyl)guanine
Homo sapiens
-
-
0.00009 - 1.2
O6-benzylguanine
0.0003
O6-furfurylguanine
Homo sapiens
-
-
0.000018
O6-thenylguanine
Homo sapiens
-
-
0.0085
O6-[(1-methyl-1H-imidazol-5-yl)methyl]guanine
Homo sapiens
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7.6
-
pH 6.0: about 80% of maximal activity, pH 7.6: about 40% of maximal activity
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 40
-
about 75% of maximal activity at 15 or at 40C
37 - 70
-
about 50% of maximal activitya at 37C and at 70C
50 - 100
-
activity increases from 50C to 100%, 60C: about 45% of the activity at 100C, 50% about 15% of maximal activity
60 - 100
-
activity at 60C is about 35% of the activity at 100C
65 - 85
-
65C: about 50% of maximal activity, 85C: about 45% of maximal activity
80 - 100
80C: optimum, 100C: substantial repair activity at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
AGT expression is increased in breast cancer compared to normal breast
Manually annotated by BRENDA team
-
activity is significantly decreased in samples from current smokers compared to nonsmokers
Manually annotated by BRENDA team
-
activity in cells adapted by exposure to N-methyl-N1-nitroguanidine, no activity in extracts of nonadapted cells
Manually annotated by BRENDA team
-
esophageal squamous cell carcinoma
Manually annotated by BRENDA team
-
no statistically significant difference in the amount of enzyme between tumors with and without activated H1-ras oncogene
Manually annotated by BRENDA team
-
D283 MED, D341 MED and Daoy
Manually annotated by BRENDA team
-
; three lines with different MGMT activity and functional status of the mismatch repair system
Manually annotated by BRENDA team
-
low 6-O-methylguanine-DNA methyltransferase activity may contribute to the chemosensitivity of some human oligodendrogliomas
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17040
amino acid sequence
19000
-
gel filtration, glycerol density gradient sedimentation
21000
-
SDS-PAGE
51000
-
enhanced green fluorescent protein-tagged enzyme, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
-
structure analysis by multinuclear multidimensional NMR spectroscopy, a two-domain protein with an alpha/beta fold, N-terminal domain consists of a three-stranded antiparallel beta sheet, N-terminus is somewhat less well defined than the C-terminus, structure is similar to homologs from other organisms that have been determined by crystallography, with some variation in the N-terminal domain, whereas the C-terminal domain is more highly conserved in both sequence and structure
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cys acceptor site is buried in the protein and, in order for reaction with a DNA substrate to take place, a change in conformation of either the substrate or the protein must occur, Glu-172 and Asn-137 play a major structural role in the AGT protein
-
hanging drop vapor diffusion method, for wild type, enzyme using 0.2 M ammonium acetate, 22% (w/v) PEG 3350, and 0.1 M HEPES pH 7.5, while for mutant enzymes R37K and Y139F using 0.1 M HEPES pH 7.5, 4% polyethylene glycol 8000, and either 4% or 8% ethylene glycol, respectively
-
sitting drop vapor diffusion method, using 0.1 M HEPES (pH 7.5), and 6% (w/v) polyethylene glycol 8000
-
crystals formed at 20C by conventional hanging drop-vapor diffusion method
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75
-
60 min, 25% loss of activity
90
-
stable for at least 30 min. 66% loss of activity after 60 min
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is denatured by 8 M urea at pH 7.5 for 30 s or by 6 M guanidine hydrochloride at pH 7.5 for 16-24 h
-
guanidine-HCl, half-time for unfolding at 30C is 52 days. An ion-pair network plays a key role in the slow unfolding
-
very stable to proteases attack and detergents such as Triton X-100
zinc provides conformational stability to hAGT that may influence its regulation
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2,2,2-trifluoroethanol
-
enzyme retains its native structure at high concentrations
2-propanol
-
enzyme retains its native structure at high concentrations
Ethanol
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enzyme retains its native structure at high concentrations
Methanol
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enzyme retains its native structure at high concentrations
SDS
-
enzyme retains its native structure at high concentrations
urea
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5 mM, melting temperature of wild-type enzyme: 91.5 C, melting temperature of mutant enzyme E83A: 89.2C, melting temperature of mutant enzyme E93A: 85.5C, melting temperature of mutant enzyme E158A: 90.6C, melting temperature of mutant enzyme E159A: 91.7 C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C stable for years
4C, stored at, storage at -20C or at -80C results in protein precipitation
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amylose resin column chromatography, Toyopearl-SuperQ column chromatography, ammonium sulfate precipitation, and Toyopearl-ether 650M column chromatography
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by immobilized metal affinity chromatography
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DEAE-Sepharose column chromatography
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expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allows a single-step isolation and purification by metal-affinity chromatography
heterologous expression by His-tag chromatography affinity
HiTrapQ column chromatography, MonoQ column chromatography, HiTrap heparin column chromatography, and Superdex 200 gel filtration
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immobilized metal affinity column chromatography
mutant V139F
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mutants purified by either nickel or glutathione affinity chromatography
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Ni-NTA resin column chromatography, gel filtration
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recombinant enzyme
Superdex 200 gel filtration
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to apparent homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA is cloned into a pET-11a plasmid and expressed in Escherichia coli
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expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allows a single-step isolation and purification by metal-affinity chromatography
expressed in CHO-9 cells
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expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL cells
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expressed in Escherichia coli Rosetta(DE3) cells
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expressed in Escherichia coli XL-1 Blue cells
expressed in Escherichia coliKT233 and in HeLa cells
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expressed in HEK-293T cells as enhanced green fluorescent protein-tagged enzyme
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expressed in U-87MG cells
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expression in CHO cells
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expression in Escherichia coli
expression in Escherichia coli strain AB1157 and isogenic strain GWR111
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expression in Escherichia coli TRG8
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expression in Escherichia coli, transgenic overexpression in mouse skin, expression in CHO cells
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expression of N-hAGT and C-hAGT in Escherichia coli XL1-Blue cells. N-hAGT, C-hAGT and full length AGT expressed from the pQE-30 vector in Escherichia coli GWR-109
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ligated into the pHybLex/Zeo plasmid and electroporated into Escherichia coli XL1-Blue, yielding a library of at least 320000 independent clones, genes of selected AGT mutants subcloned into the pGEX-2T plasmid, allowing for the expression of the AGT mutants as GST fusion proteins
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mutants subcloned into pGEX-2T for expression as a GSTAGT fusion, or into pET15b for expression as a 6 x HisAGT fusion protein
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overexpressed in Escherichia coli strain UC978
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overexpression in CHO-derived Tet-on-inducible cells, knockdown of MGMT expression with small interfering RNA in HONE-1 cells
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recombinant AGT and mutants expressed in Escherichia coli, pQE expression vectors for wild-type and mutant AGT production and plasmid pREP4 cotransformed into TRG8 cells to suppress basal AGT protein expression
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recombinant variants are expressed in HMS174 pLyS cells
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the gene is placed under the control of the lac promoter and overexpressed in the methyltransferase-deficient Escherichia coli strain KT233
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transformed into Escherichia coli BL21(DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
a loss of MGMT expression is noted in 20 cases of 60 total soft tissue sarcomas
-
ependymomas lack MGMT promoter hypermethylation and show high MGMT protein expression
-
extensive methylation of MGMT promoter is associated with loss/reduced protein expression, methylation of MGMT promoter region is essential for methylation-induced silencing of this gene
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interleukin-24 down-regulates O6-methylguanine-DNA methyltransferase in melanoma cells, accumulation of functional p53 is essential for interleukin-24-induced down-regulation of O6-methylguanine-DNA methyltransferase
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MGMT activity is significantly higher in the group with the lowest consumption of vegetables than in the group with the greatest vegetable consumption
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neither short-term (24 h) nor long-term changes (7 weeks) in S-adenosyl-L-methionine/S-adenosylhomocysteine ratio alter global or MGMT promoter methylation, however, experimentally elevated S-adenosylhomocysteine levels significantly decrease MGMT mRNA levels by more than 50%
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promoter methylation of O6-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53
-
resistance to alkylating agents such as temozolomide correlates with increased expression of O6-methylguanine-DNA methyltransferase
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there is a good correlation between the increasing MGMT promoter methylation and decreased MGMT expression, in most patients who has been treated with temozolomide, there is decreased MGMT expression in the post treatment sample when compared to the primary sample
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V139F
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has an increased ability to protect against the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine
A121E
-
no major structural change upon energy minimization, is within the DNA binding region and may therefore affect DNA binding of MGMT
A121T
-
no major structural change upon energy minimization, is within the DNA binding region and may therefore affect DNA binding of MGMT
A154T
-
ca. 4fold increased activity, AGT mutant selected using phage display
A41D/S115T/I151N
-
1.6fold increased activity, AGT mutant selected using yeast three-hybrid system
C145F
-
appears to cause a similar change in the AGT structure as alkylation of the active site and provides a model for detailed study of the mechanism of degradation
C145S
-
inactive mutant enzyme forms a specific and stable complex with a 6-O-methylguanine-containing oligonucleotide substrate
C24A
-
mutation of Cys24 prevents the zinc-dependent alkyl transferase by N-terminal domain human AGT
D42E/A51T/A64V/K104M
-
0.5fold increased activity, AGT mutant selected using yeast three-hybrid system
D42E/P47L/V155L/K178M
-
1.9fold increased activity, AGT mutant selected using yeast three-hybrid system
E110D/L120M
-
0.7fold increased activity, AGT mutant selected using yeast three-hybrid system
E166D
-
no major structural change upon energy minimization
E25K
-
0.7fold increased activity, AGT mutant selected using yeast three-hybrid system
E92D/I151V/R175W
-
1.0fold increased activity, AGT mutant selected using yeast three-hybrid system
F79I/V88I/F89L
-
0.7fold increased activity, AGT mutant selected using yeast three-hybrid system
G122C
-
0.9fold increased activity, AGT mutant selected using yeast three-hybrid system
G132R
-
no major structural change upon energy minimization, is within the DNA binding region and may therefore affect DNA binding of MGMT
G156C
-
mutant form expressed by the medulloblastoma cell line D283 MED, mutant enzyme is not easily inhibited by O6-benzylguanine
G160W
-
cells overexpressing W160AGT do not become labeled, even when incubated with O6-propargylguanine for extended periods of time
H29A
-
does not show the stability enehancement of wild-type hAGT with its intact zinc coordination sphere
H71Y/A154T
-
3.3fold increased activity, AGT mutant selected using phage display
H85A
-
does not show the stability enehancement of wild-type hAGT with its intact zinc coordination sphere
I143V/K178R
K104E/T127A/A154T
-
4.5fold increased activity, AGT mutant selected using phage display
K107L
-
mutant is deficient in DNA repair
K107R/A154T
-
4.7fold increased activity, AGT mutant selected using phage display
K165R
-
does not abolish activity on 6-O-methylguanine but greatly reduces the ability to react with O6-benzylguanine
K165T
-
mutant form expressed by the medulloblastoma cell line D341 MED, mutant enzyme is not easily inhibited by O6-benzylguanine
K8R/K104E/I151T
-
1.4fold increased activity, AGT mutant selected using phage display
K8T/A51T/I112V/A154T
-
5.5fold increased activity, AGT mutant selected using phage display
K8T/T127A/A154T/H174R
-
5.8fold increased activity, AGT mutant selected using phage display
L33F/A68T
-
2.1fold increased activity, AGT mutant selected using yeast three-hybrid system
L33F/N123Y
-
2.2fold increased activity, AGT mutant selected using yeast three-hybrid system
L33F/V44A/V52A/A154T
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6.1fold increased activity, AGT mutant selected using phage display
L66M/K131R
-
1.5fold increased activity, AGT mutant selected using yeast three-hybrid system
L84F/I143V/K178R
-
enhanced green fluorescent protein-tagged MGMT variants exhibit nuclear localization patterns indistinguishable from wild type enzyme, upon exposure to O6-benzylguanine, the L84F/I143V/K178R variant is degraded more rapidly than wild type
M1V/V164M
-
1.9fold increased activity, AGT mutant selected using phage display
N123S
-
1.3fold increased activity, AGT mutant selected using phage display
N123V
-
no major structural change upon energy minimization, is within the DNA binding region and may therefore affect DNA binding of MGMT
N150D
-
1.8fold increased activity, AGT mutant selected using phage display
N157/S159/C62A/C150N/G131K/G132T/M134L/R135S/Q115S/Q116H/K125A/A127T/R128A/S151I/S152N
-
called MAGT with 15 different mutations in a single protein, has23fold increase in activity relative to wild-type, is resistant against N9-substituted BG derivatives used for inhibition of wild-type, shows suppressed affinity towards DNA
P140A
-
70% reduced ability of the protein to react with Br(CH2)2Br
Q90R/K101N/F108I/V164L
-
1.2fold increased activity, AGT mutant selected using yeast three-hybrid system
R128A
-
substantially reduced AGT-mediated increase in toxicity and the induction of mutations in Escherichia coli cells treated with Br(CH2)2Br, is able to react with Br(CH2)2Br at the Cys145 acceptor site, but the resulting AGT-Cys145S-(CH2)2Br is much less able to produce a covalent adduct with DNA
R128G
-
reduces the AGT repair efficiency, no smaller effects with the O6-benzylguanine substrate than the 6-O-methylguanine
R128L
-
reduces the AGT repair efficiency, smaller effects with the O6-benzylguanine substrate than the 6-O-methylguanine
R175L
-
0.6fold increased activity, AGT mutant selected using yeast three-hybrid system
T11I/N67K/Q72L
-
1.0fold increased activity, AGT mutant selected using yeast three-hybrid system
T127A
-
2.2fold increased activity, AGT mutant selected using phage display
T38M/A41D/A64T/G173C
-
0.8fold increased activity, AGT mutant selected using yeast three-hybrid system
V149I/A154T
-
4.5fold increased activity, AGT mutant selected using phage display
V44G/V106A/I151T/A170T
-
1.4fold increased activity, AGT mutant selected using yeast three-hybrid system
V46A/A50V/P58V/A154T
-
3.4fold increased activity, AGT mutant selected using phage display
V52A/I151S/K178E
-
2.3fold increased activity, AGT mutant selected using phage display
V52I/V164M
-
2.1fold increased activity, AGT mutant selected using yeast three-hybrid system
W65C
-
no major structural change upon energy minimization, possibly unstable
Y114A
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reduced ability of the protein to react with Br(CH2)2Br as measured by loss of activity
Y114E
-
substantially reduced AGT-mediated increase in toxicity and the induction of mutations in Escherichia coli cells treated with Br(CH2)2Br
P140K
-
mutant confers resistance to N,N'-bis(2-chloroethyl)-N-nitrosourea and O(6)-benzylguanine
R37E
-
the mutant exhibits a 5fold lower affinity for the methylated duplex DNA compared to the wild type enzyme
R37K
-
the variant performs a sub-optimal alkylated-DNA repair in vitro compared to the wild type enzyme
T15S
-
the mutant exhibits a 2fold-lower affinity for double stranded methylated DNA compared to the wild type enzyme
Y139F
-
the mutant exhibits a 10fold lower affinity for the methylated duplex DNA compared to the wild type enzyme
E158A
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melting temperature of mutant enzyme E83A at 5 mM urea is 90.6C, compared to 91.5C for the wild-type enzyme
E159A
-
melting temperature of mutant enzyme E83A at 5 mM urea is 91.7C, compared to 91.5C for the wild-type enzyme
E83A
-
melting temperature of mutant enzyme E83A at 5 mM urea is 89.2C, compared to 91.5C for the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
renaturation by dilution with buffer from 8 M urea or 6 M guanidine HCl instantly restores the secondary structure of the Ada protein to a state that is almost indistinguishable from the native state. Kinetics of renaturation is fast, at 0C the restoration is quantitative in less than 30 s. The heat-coagulated protein can be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
diagnostics
-
introduction of the MS-MLPA assay may not only be helpful for predicting response of gliomas to temozolomide, but may also facilitate tailor-made treatment with other chemotherapeutic agents for a variety of tumors
drug development
-
highly effective inactivation of MGMT by an oligodeoxyribonucleotide containing O6-(4-bromothenyl)guanine suggests that such oligodeoxyribonucleotides might have therapeutic applications if problems of delivery can be addressed
medicine
molecular biology
-
modest binding cooperativity and high binding densities of AGT are adaptations that allow the enzyme to efficiently search for lesions in the context of chromatin remodeling and DNA replication
Show AA Sequence (7623 entries)
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