Literature summary for 2.1.1.56 extracted from

Fabrega, C.; Hausmann, S.; Shen, V.; Shuman, S.; Lima, C.D.
Structure and mechanism of mRNA cap (guanine-N7) methyltransferase (2004), Mol. Cell, 13, 77-89.

Cloned(Commentary)

Cloned (Comment)	Organism
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	Encephalitozoon cuniculi

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Encephalitozoon cuniculi

Crystallization (Comment)

Organism

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

Encephalitozoon cuniculi

Protein Variants

Protein Variants	Comment	Organism
D122A/S123A	site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, only slightly at 37°C	Encephalitozoon cuniculi
D78A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 30°C and 37°C, but at 25°C	Encephalitozoon cuniculi
D94A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo	Encephalitozoon cuniculi
F141A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo	Encephalitozoon cuniculi
I95A	site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C, 30°C, and 37°C, no effect on enzyme function	Encephalitozoon cuniculi
I95A/Y124A	site-directed mutagenesis, only slight complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, growth defects	Encephalitozoon cuniculi
K54A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, inactive mutant in vivo	Encephalitozoon cuniculi
K75A	site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype	Encephalitozoon cuniculi
K81A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo	Encephalitozoon cuniculi
L216A	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	Encephalitozoon cuniculi
N50A	site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C, only modest growth defects	Encephalitozoon cuniculi
N50A/Y284A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo	Encephalitozoon cuniculi
N51A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C	Encephalitozoon cuniculi
R106A	site-directed mutagenesis, only slight complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C	Encephalitozoon cuniculi
R47A	site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C	Encephalitozoon cuniculi
R47A/K75A	site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo	Encephalitozoon cuniculi
Y124A	site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype	Encephalitozoon cuniculi
Y284A	site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, slow growth phenotype	Encephalitozoon cuniculi

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
S-adenosyl-L-methionine + G(5')pppR-RNA	Encephalitozoon cuniculi	essential and defining step in the eukaryotic mRNA synthesis	S-adenosyl-L-homocysteine + m7G(5')pppR-RNA	-	?

Organism

Organism	UniProt	Comment	Textmining
Encephalitozoon cuniculi	-	microsporidian parasite, enzyme Ecm1	-

Purification (Commentary)

Purification (Comment)	Organism
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	Encephalitozoon cuniculi

Reaction

Reaction	Comment	Organism	Reaction ID
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	Encephalitozoon cuniculi	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
S-adenosyl-L-methionine + G(5')pppR-RNA	essential and defining step in the eukaryotic mRNA synthesis	Encephalitozoon cuniculi	S-adenosyl-L-homocysteine + m7G(5')pppR-RNA	-	?
S-adenosyl-L-methionine + G(5')pppR-RNA	enzyme contains the conserved S-adenosyl-L-methionine-binding motif VLDLGXGXG, structural features for cap guanine specificity	Encephalitozoon cuniculi	S-adenosyl-L-homocysteine + m7G(5')pppR-RNA	-	?

Subunits

Subunits	Comment	Organism
More	primary sequence, sequence comparison with other species, enzyme-ligand structures	Encephalitozoon cuniculi

Synonyms

Synonyms	Comment	Organism
mRNA cap (guanine-N7) methyltransferase	-	Encephalitozoon cuniculi