Literature summary for 2.1.1.56 extracted from

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 (2006), J. Biol. Chem., 281, 35904-35913.

Application

Application	Comment	Organism
analysis	cap methylation is a principal target of the antifungal activity of sinefungin	Encephalitozoon cuniculi
drug development	cap methylation is a principal target of the antifungal activity of sinefungin	Saccharomyces cerevisiae

Cloned(Commentary)

Cloned (Comment)	Organism
as a His6-Smt3 fusion protein in Escherichia coli BL21(DE3) CodonPlus RIL using a pET-based pSmt3-TOPO vector	Saccharomyces cerevisiae
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)	Encephalitozoon cuniculi

Crystallization (Commentary)

Crystallization (Comment)	Organism
Ecm1-sinefungin binary complex, by vapor diffusion, to 2.6 A resolution	Encephalitozoon cuniculi

Protein Variants

Protein Variants	Comment	Organism
D70A	lethal	Encephalitozoon cuniculi
D70E	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
D70N	lethal	Encephalitozoon cuniculi
D78A	lethal	Encephalitozoon cuniculi
D78E	no growth at 37°C	Encephalitozoon cuniculi
D78N	lethal	Encephalitozoon cuniculi
D94A	lethal	Encephalitozoon cuniculi
D94E	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
D94N	lethal	Encephalitozoon cuniculi
E225A	no growth at 37°C	Encephalitozoon cuniculi
F141A	lethal	Encephalitozoon cuniculi
F141H	grows well at 25 and 30°C but forms pinpoint colonies at 37°C	Encephalitozoon cuniculi
F141I	grows well at 25 and 30°C but forms pinpoint colonies at 37°C	Encephalitozoon cuniculi
F141L	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
F141N	lethal	Encephalitozoon cuniculi
F141V	grows well at 25 and 30°C but forms pinpoint colonies at 37°C	Encephalitozoon cuniculi
F214A	no growth at 37°C	Encephalitozoon cuniculi
H144A	no effect on yeast growth at 25 or 30°C, although it slowly grows at 37°C	Encephalitozoon cuniculi
H144A/Y145F	fully functional	Encephalitozoon cuniculi
H144A/Y145L	defective in vivo, fails to grow at 37°C, forms microcolonies at 30°C, and grows slowly at 25°C	Encephalitozoon cuniculi
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	Encephalitozoon cuniculi
K54A	lethal	Encephalitozoon cuniculi
K54Q	lethal	Encephalitozoon cuniculi
K54R	lethal	Encephalitozoon cuniculi
K75A	no growth at 37°C	Encephalitozoon cuniculi
K81A	lethal	Encephalitozoon cuniculi
K81Q	lethal	Encephalitozoon cuniculi
K81R	lethal	Encephalitozoon cuniculi
N51A	no growth at 37°C	Encephalitozoon cuniculi
N51D	lethal	Encephalitozoon cuniculi
P175A	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
R106A	lethal	Encephalitozoon cuniculi
R106K	lethal	Encephalitozoon cuniculi
R106Q	lethal	Encephalitozoon cuniculi
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	Encephalitozoon cuniculi
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	Encephalitozoon cuniculi
Y124A	no growth at 37°C	Encephalitozoon cuniculi
Y145A	lethal	Encephalitozoon cuniculi
Y145F	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
Y145H	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
Y145I	intermediate phenotype of slow growth at 25 and 30°C and microcolony formation at 37°C	Encephalitozoon cuniculi
Y145L	grows as well as the wild-type ECM1 strain at all temperatures	Encephalitozoon cuniculi
Y145S	lethal	Encephalitozoon cuniculi
Y145V	lethal	Encephalitozoon cuniculi
Y212A	no growth at 37°C	Encephalitozoon cuniculi

Inhibitors

Inhibitors	Comment	Organism
S-adenosyl-L-homocysteine	inhibits in a concentration-dependent fashion in the presence of 0.025 mM [3H-CH3]S-adenosyl-L-methionine	Saccharomyces cerevisiae
sinefungin	inhibits Ecm1 with modest potency	Encephalitozoon cuniculi
sinefungin	extremely potent inhibitor, binds 900fold more avidly than S-adenosylhomocysteine or S-adenosylmethionine, sensitivity to growth inhibition is diminished when Abd1 is overexpressed	Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.006	-	S-adenosyl-L-methionine	-	Saccharomyces cerevisiae
0.24	-	GpppA	-	Saccharomyces cerevisiae

Organism

Organism	UniProt	Comment	Textmining
Encephalitozoon cuniculi	Q8SR66	-	-
Saccharomyces cerevisiae	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
His6-Smt3-Abd1 by nickel-nitrilotriacetic acid fast flow column chromatography, Abd1 free of the tag by gel filtration, to homogeneity	Saccharomyces cerevisiae
recombinant Ecm1 mutant proteins, by nickel-agarose chromatography	Encephalitozoon cuniculi

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
S-adenosyl-L-methionine + GpppA	-	Saccharomyces cerevisiae	S-adenosyl-L-homocysteine + m7GpppA	-	?
S-adenosyl-L-methionine + GpppA	-	Encephalitozoon cuniculi	S-adenosyl-L-homocysteine + m7GpppA	-	?

Synonyms

Synonyms	Comment	Organism
Abd1	-	Saccharomyces cerevisiae
Ecm1	-	Encephalitozoon cuniculi
mRNA cap (guanine-N7) methyltransferase	-	Saccharomyces cerevisiae
mRNA cap (guanine-N7) methyltransferase	-	Encephalitozoon cuniculi

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
25	37	-	Encephalitozoon cuniculi

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.0983	-	GpppA	-	Saccharomyces cerevisiae

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.000021	-	-	Saccharomyces cerevisiae	sinefungin
0.021	-	-	Saccharomyces cerevisiae	S-adenosyl-L-homocysteine