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Information on EC 2.1.1.56 - mRNA (guanine-N7)-methyltransferase and Organism(s) Encephalitozoon cuniculi and UniProt Accession Q8SR66
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2.1.1.56 mRNA (guanine-N7)-methyltransferaseIUBMB Comments
The terminal N7-methylguanosine facilitates gene expression in eukaryotic cells and is recognized by cap-binding proteins.
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Word Map
- 2.1.1.56
- dengue
- viruses
- influenza
- flavivirus
- pandemic
-
sars-cov
- coronaviruses
- exoribonuclease
- encephalitis
- mtases
- covid-19
-
anti-ns1
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guanylyltransferase
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replicase
-
proofreading
-
mosquito-borne
-
denv-1
- rig-i
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arthropod-borne
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anti-denv
- coronaviridae
-
chikv
- sinefungin
- remdesivir
- gpppg
-
ns1-specific
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anti-dengue
- bluetongue
- medicine
- analysis
- drug development
- synthesis
The taxonomic range for the selected organisms is: Encephalitozoon cuniculi
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
non-structural protein 1, nsp14, cap methyltransferase, n7-mtase, mrna cap methyltransferase, nonstructural protein 14, tbcmt1, n7-methyltransferase, tbcgm1, guanine-7-methyltransferase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
guanine-7-methyltransferase
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messenger ribonucleate guanine 7-methyltransferase
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messenger RNA guanine 7-methyltransferase
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methyltransferase, messenger ribonucleate guanine 7-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + a 5'-(5'-triphosphoguanosine)-[mRNA] = S-adenosyl-L-homocysteine + a 5'-(N7-methyl 5'-triphosphoguanosine)-[mRNA]
in-line mechanism of methyl transfer without direct contact between the enzyme and the N7 atom of guanine, which is the attacking nucleophile, the methyl carbon of S-adenosyl-L-methionine, or the sulfur of S-adenosyl-L-methionine/S-adenosyl-L-homocysteine, enzyme-ligand structures, active site structure
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
methyl group transfer
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:mRNA (guanine-N7)-methyltransferase
The terminal N7-methylguanosine facilitates gene expression in eukaryotic cells and is recognized by cap-binding proteins.
CAS REGISTRY NUMBER
COMMENTARY
56941-25-4
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + dGTP
S-adenosyl-L-homocysteine + m7dGTP
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-
-
?
S-adenosyl-L-methionine + GDP
S-adenosyl-L-homocysteine + m7GDP
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-
-
?
S-adenosyl-L-methionine + GTP
S-adenosyl-L-homocysteine + m7GTP
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-
-
?
S-adenosyl-L-methionine + GpppA
S-adenosyl-L-homocysteine + m7GpppA
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-
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?
S-adenosyl-L-methionine + GpppA
S-adenosyl-L-homocysteine + m7GpppA
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-
-
-
?
S-adenosyl-L-methionine + G(5')pppR-RNA
S-adenosyl-L-homocysteine + m7G(5')pppR-RNA
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essential and defining step in the eukaryotic mRNA synthesis
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?
S-adenosyl-L-methionine + G(5')pppR-RNA
S-adenosyl-L-homocysteine + m7G(5')pppR-RNA
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enzyme contains the conserved S-adenosyl-L-methionine-binding motif VLDLGXGXG, structural features for cap guanine specificity
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?
additional information
?
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does not methylate ATP, CTP, UTP, ITP, or m7GTP
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?
additional information
?
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does not methylate ATP, CTP, UTP, ITP, or m7GTP
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + G(5')pppR-RNA
S-adenosyl-L-homocysteine + m7G(5')pppR-RNA
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essential and defining step in the eukaryotic mRNA synthesis
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
S-adenosyl-L-homocysteine
inhibits methylation of GTP in the presence of 0.005 mM S-adenosyl-L-methionine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 7.5
methyl transfer by Ecm1 declines sharply below pH 6.0
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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primary sequence, sequence comparison with other species, enzyme-ligand structures
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ecm1-aza-S-adenosyl-L-methionine binary complex, by vapor diffusion, reveals that the inhibitor occupies the same site as S-adenosyl-L-methionine
Ecm1-sinefungin binary complex, by vapor diffusion, to 2.6 A resolution
6.5 mg/ml purified native or selenomethionine-labeled, or Hg-labeled enzyme in complex with S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, or the cap guanylate plus S-adenosyl-L-homocysteine, in 20 mM Tris-HCl, pH 8.0, 220 mM NaCl, vapour diffusion against a well solution of 1.2 M sodium potassium tartrate, 50 mM bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, pH 6.0-6.2, and 20 mM DTT, X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, modeling
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D70A
D70E
D70N
D78A
D78E
D78N
D94A
D94E
D94N
E225A
F141A
F141H
grows well at 25 and 30°C but forms pinpoint colonies at 37°C
F141I
grows well at 25 and 30°C but forms pinpoint colonies at 37°C
F141L
grows as well as the wild-type ECM1 strain at all temperatures
F141V
grows well at 25 and 30°C but forms pinpoint colonies at 37°C
F214A
H144A
H144A/Y145L
defective in vivo, fails to grow at 37°C, forms microcolonies at 30°C, and grows slowly at 25°C
K267A
K54A
K54Q
K54R
K81Q
K81R
N51D
P175A
grows as well as the wild-type ECM1 strain at all temperatures
R106A
R106K
R106Q
R59A
R84A
Y145A
Y145F
Y145H
grows as well as the wild-type ECM1 strain at all temperatures
Y145I
intermediate phenotype of slow growth at 25 and 30°C and microcolony formation at 37°C
Y145L
Y145S
Y212A
D122A/S123A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, only slightly at 37°C
D78A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 30°C and 37°C, but at 25°C
D94A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
F141A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
I95A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C, 30°C, and 37°C, no effect on enzyme function
I95A/Y124A
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site-directed mutagenesis, only slight complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, growth defects
K54A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, inactive mutant in vivo
K75A
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site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype
K81A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
L216A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C, no effect on cell growth
N50A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C, only modest growth defects
N50A/Y284A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
N51A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C
R106A
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site-directed mutagenesis, only slight complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C
R47A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C
R47A/K75A
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site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
Y124A
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site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype
Y284A
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site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, slow growth phenotype
additional information
D70A
lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
D70E
grows as well as the wild-type ECM1 strain at all temperatures
D78A
lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
D94A
suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
D94E
grows as well as the wild-type ECM1 strain at all temperatures
F141A
suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
H144A
no effect on yeast growth at 25 or 30°C, although it slowly grows at 37°C
K267A
grows as well as the wild-type ECM1 strain at all temperatures and is as active as wild-type Ecm1 in GpppA methylation in vitro
K54A
lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
R59A
grows as well as the wild-type ECM1 strain at all temperatures and is as active as wild-type Ecm1 in GpppA methylation in vitro
R84A
grows as well as the wild-type ECM1 strain at all temperatures and is as active as wild-type Ecm1 in GpppA methylation in vitro
Y145F
grows as well as the wild-type ECM1 strain at all temperatures
Y145L
grows as well as the wild-type ECM1 strain at all temperatures
additional information
residues Lys-54, Asp-70, Asp-78, and Asp-94 that comprise the S-adenosyl-L-methionine-binding pocket are all essential for Ecm1 function in yeast, i.e. alanine substitutions at each of these positions are lethal, loss of function caused by the elimination of Phe-141 attests to the importance of its van der Waals interactions with the guanosine methyl acceptor seen in the Ecm1-GTP cocrystal
additional information
-
residues Lys-54, Asp-70, Asp-78, and Asp-94 that comprise the S-adenosyl-L-methionine-binding pocket are all essential for Ecm1 function in yeast, i.e. alanine substitutions at each of these positions are lethal, loss of function caused by the elimination of Phe-141 attests to the importance of its van der Waals interactions with the guanosine methyl acceptor seen in the Ecm1-GTP cocrystal
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant Ecm1 mutant proteins, by nickel-agarose chromatography
recombinant N-terminally His6-tagged enzyme as Smt3-fusion protein from Escherichia coli strain BL21(DE3), removal of His-Smt3 tag by specific protease Ulp1, purification by metal affinity chromatography and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
inserted into vector pET16b and transformed into Escherichia coli BL21(DE3), yeast strain YBS40 transformed with CEN TRP1 plasmids containing the wild-type and mutant alleles of ECM1
yeast strain YBS40 transformed with CEN TRP1 plasmids containing the wild-type and mutant alleles of ECM1, the pET16-Ecm1 plasmids introduced into Escherichia coli BL21(DE3)
expression of N-terminally His6-tagged enzyme, residues 1-298, as Smt3-fusion protein in Escherichia coli strain BL21(DE3) or strain B834DE3, expression of wild-type and mutant alleles under control of a yeast promotor in a Saccharomyces cerevisiae abd1DELTA null mutant strain
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
cap methylation is a principal target of the antifungal activity of sinefungin
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Fabrega, C.; Hausmann, S.; Shen, V.; Shuman, S.; Lima, C.D.
Structure and mechanism of mRNA cap (guanine-N7) methyltransferase
Mol. Cell
13
77-89
2004
Encephalitozoon cuniculi
Hausmann, S.; Zheng, S.; Fabrega, C.; Schneller, S.W.; Lima, C.D.; Shuman, S.
Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis
J. Biol. Chem.
280
20404-20412
2005
Encephalitozoon cuniculi (Q8SR66), Encephalitozoon cuniculi
Zheng, S.; Hausmann, S.; Liu, Q.; Ghosh, A.; Schwer, B.; Lima, C.D.; Shuman, S.
Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungins antifungal activity
J. Biol. Chem.
281
35904-35913
2006
Saccharomyces cerevisiae, Encephalitozoon cuniculi (Q8SR66), Encephalitozoon cuniculi
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