Cloned (Comment) | Organism |
---|---|
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 | Encephalitozoon cuniculi |
Crystallization (Comment) | Organism |
---|---|
Ecm1-aza-S-adenosyl-L-methionine binary complex, by vapor diffusion, reveals that the inhibitor occupies the same site as S-adenosyl-L-methionine | Encephalitozoon cuniculi |
Protein Variants | Comment | Organism |
---|---|---|
D70A | lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity | Encephalitozoon cuniculi |
D70E | growth at all temperatures | Encephalitozoon cuniculi |
D70N | lethal | Encephalitozoon cuniculi |
D78A | lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity | Encephalitozoon cuniculi |
D78E | growth at all temperatures except at 37°C | Encephalitozoon cuniculi |
D78N | growth at all temperatures | Encephalitozoon cuniculi |
D94A | suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity | Encephalitozoon cuniculi |
D94E | growth at all temperatures | Encephalitozoon cuniculi |
D94N | lethal | Encephalitozoon cuniculi |
E225A | growth at all temperatures except at 37°C | Encephalitozoon cuniculi |
E225D | growth at all temperatures | Encephalitozoon cuniculi |
E225Q | growth at all temperatures | Encephalitozoon cuniculi |
F141A | suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity | Encephalitozoon cuniculi |
F214A | growth at all temperatures except at 37°C | Encephalitozoon cuniculi |
F214L | growth at all temperatures, slow growth at 37°C | Encephalitozoon cuniculi |
H144A | growth at all temperatures | Encephalitozoon cuniculi |
K267A | growth at all temperatures | Encephalitozoon cuniculi |
K54A | lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity | Encephalitozoon cuniculi |
K54Q | lethal | Encephalitozoon cuniculi |
K54R | lethal | Encephalitozoon cuniculi |
K75Q | slow growth at all temperatures | Encephalitozoon cuniculi |
K75R | growth at all temperatures | Encephalitozoon cuniculi |
K81Q | lethal | Encephalitozoon cuniculi |
K81R | lethal | Encephalitozoon cuniculi |
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 | Encephalitozoon cuniculi |
N51D | lethal | Encephalitozoon cuniculi |
R106A | lethal | Encephalitozoon cuniculi |
R106K | lethal | Encephalitozoon cuniculi |
R106Q | lethal | Encephalitozoon cuniculi |
R59A | growth at all temperatures | Encephalitozoon cuniculi |
R84A | growth at all temperatures | Encephalitozoon cuniculi |
Y124F | growth at all temperatures, slow growth at 37°C | Encephalitozoon cuniculi |
Y145A | lethal | Encephalitozoon cuniculi |
Y145F | growth at all temperatures | Encephalitozoon cuniculi |
Y145L | growth at all temperatures | Encephalitozoon cuniculi |
Y145S | lethal | Encephalitozoon cuniculi |
Y212A | growth at all temperatures except at 37°C | Encephalitozoon cuniculi |
Y212F | growth at all temperatures | Encephalitozoon cuniculi |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
aza-S-adenosyl-L-methionine | - |
Encephalitozoon cuniculi | |
carbocyclic aza-S-adenosyl-L-methionine | - |
Encephalitozoon cuniculi | |
S-adenosyl-L-homocysteine | inhibits methylation of GTP in the presence of 0.005 mM S-adenosyl-L-methionine | Encephalitozoon cuniculi | |
sinefungin | - |
Encephalitozoon cuniculi |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.1 | - |
GpppA | - |
Encephalitozoon cuniculi | |
1 | - |
GTP | - |
Encephalitozoon cuniculi | |
2.4 | - |
GDP | - |
Encephalitozoon cuniculi |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
40000 | - |
1 * 40000, SDS-PAGE | Encephalitozoon cuniculi |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Encephalitozoon cuniculi | Q8SR66 | - |
- |
Purification (Comment) | Organism |
---|---|
by nickel-affinity chromatography | Encephalitozoon cuniculi |
Storage Stability | Organism |
---|---|
-80 °C, Tris-HCl buffer, pH 7.5, 10% glycerol | Encephalitozoon cuniculi |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | does not methylate ATP, CTP, UTP, ITP, or m7GTP | Encephalitozoon cuniculi | ? | - |
? | |
S-adenosyl-L-methionine + dGTP | - |
Encephalitozoon cuniculi | S-adenosyl-L-homocysteine + m7dGTP | - |
? | |
S-adenosyl-L-methionine + GDP | - |
Encephalitozoon cuniculi | S-adenosyl-L-homocysteine + m7GDP | - |
? | |
S-adenosyl-L-methionine + GpppA | - |
Encephalitozoon cuniculi | S-adenosyl-L-homocysteine + m7GpppA | - |
? | |
S-adenosyl-L-methionine + GTP | - |
Encephalitozoon cuniculi | S-adenosyl-L-homocysteine + m7GTP | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | 1 * 40000, SDS-PAGE | Encephalitozoon cuniculi |
Synonyms | Comment | Organism |
---|---|---|
Ecm1 | - |
Encephalitozoon cuniculi |
mRNA cap (guanine N-7) methyltransferase | - |
Encephalitozoon cuniculi |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
- |
Encephalitozoon cuniculi |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
6 | 7.5 | methyl transfer by Ecm1 declines sharply below pH 6.0 | Encephalitozoon cuniculi |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | reaction is cofactor-independent | Encephalitozoon cuniculi |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.0015 | - |
- |
Encephalitozoon cuniculi | sinefungin | |
0.004 | - |
- |
Encephalitozoon cuniculi | S-adenosyl-L-homocysteine | |
0.035 | - |
- |
Encephalitozoon cuniculi | carbocyclic aza-S-adenosyl-L-methionine | |
0.1 | - |
- |
Encephalitozoon cuniculi | aza-S-adenosyl-L-methionine |