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D146A
-
mutant completely loses the N-7 activity (methylation of N-7 position of guanine) and 2'-O methylation activity. Mutation defective in N-7 methylation is lethal for DNV replication
E217A
-
mutants retains 59% of wild-type N-7 (methylation of N-7 position of guanine) activity
K181A
-
mutants retains 16% of wild-type N-7 (methylation of N-7 position of guanine) activity
K61A
-
mutants retains 34% of wild-type N-7 (methylation of N-7 position of guanine) activity
D122A/S123A
-
site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, only slightly at 37°C
E225D
growth at all temperatures
E225Q
growth at all temperatures
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
F214L
growth at all temperatures, slow growth at 37°C
H144A/Y145F
fully functional
H144A/Y145L
defective in vivo, fails to grow at 37°C, forms microcolonies at 30°C, and grows slowly at 25°C
I95A
-
site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C, 30°C, and 37°C, no effect on enzyme function
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
K75Q
slow growth at all temperatures
K75R
growth at all temperatures
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
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
N50A/Y284A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
P175A
grows as well as the wild-type ECM1 strain at all temperatures
R47A
-
site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, slightly reduced at 37°C
R47A/K75A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
Y124F
growth at all temperatures, slow growth at 37°C
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
Y212F
growth at all temperatures
Y284A
-
site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, slow growth phenotype
D330A
-
the mutation does not affect viral replication, sensitivity to mutagen, or inhibition by interferon-beta compared to the wild type
G332A
-
the mutant displays delayed replication kinetics and decreased peak titers relative to the wild type virus. In addition, replication of mutant virus is diminished following treatment of cells with interferon-beta, and mutant genomes are translated less efficiently both in vitro and during viral infection
C416R
mutation abolishes the N7-MTase activity on GTP
D273A
97% of wild-type methylation activity
D331E
70% of wild-type methylation activity
G416R
45% of wild-type methylation activity
K61A
97% of wild-type methylation activity
L419A
complete loss of methylation activity
L419R
15-50% of wild-type activity
N256A
97% of wild-type methylation activity
N334A
89% of wild-type methylation activity
N388A
83% of wild-type methylation activity
D1924A
-
the mutant shows severely reduced activity compared to the wild type enzyme
E1996A
-
the mutant shows severely reduced activity compared to the wild type enzyme
K1813A
-
the mutant shows severely reduced activity compared to the wild type enzyme
K1959A
-
the mutant shows severely reduced activity compared to the wild type enzyme
D144A
-
TbCmt1 mutant shows 5% of wild-type activity
D80A
-
TbCmt1 mutant is unable to support methylation at levels of input protein sufficient to convert all the input substrate to the methylated form by wild-type TbCmt1, specific activity is less than 0.1% of the wild-type activity
E258A
-
TbCmt1 mutant is unable to support methylation at levels of input protein sufficient to convert all the input substrate to the methylated form by wild-type TbCmt1, specific activity is less than 0.1% of the wild-type activity
F171A
-
TbCmt1 mutant displays near-wild-type activity
H174A
-
TbCmt1 mutant displays near-wild-type activity
K83A
-
TbCmt1 mutant is unable to support methylation at levels of input protein sufficient to convert all the input substrate to the methylated form by wild-type TbCmt1, specific activity is less than 0.1% of the wild-type activity
V241A
-
TbCmt1 mutant displays near-wild-type activity
V242A
-
TbCmt1 mutant displays near-wild-type activity
Y175A
-
TbCmt1 mutant shows 3% of wild-type activity
Y246A
-
TbCmt1 mutant shows 2% of wild-type activity
C173A/S174A
-
modest defect, grows well at 19, 30 and 34°C, but very slowly at 37°C
D192A/S193A
-
temperature-sensitive vD12 allele, near-lethality, only grows very slowly at 19°C
D52A/L53A
-
modest defect, grows well at 30°C, but slowly at 34°C and not at 37°C
D545A
-
the mutant has 102% of wild type activity
D598E
-
the mutant enzyme has 1% of wild type activity
D598N
-
the mutant enzyme hasless than 1% of wild type activity
D604E
-
the mutant enzyme has 112% of wild type activity
D604N
-
the mutant enzyme has less than 1% of wild type activity
D620E
-
the mutant enzyme has 59% of wild type activity
D620N
-
the mutant enzyme has 11% of wild type activity
D657A
-
the mutant enzyme has 112% of wild type activity
D676A
-
the mutant enzyme has less than 1% of wild type activity
D676E
-
the mutant enzyme has 40% of wild type activity
D676N
-
the mutant enzyme has less than 1% of wild type activity
E104A/G11A
-
modest defect, grows well at 19, 30 and 34 °C, but slowly at 37°C
E24A-25A
-
temperature-sensitive vD12 allele, does not grow at 37°C
E275A/N276A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
E763A
-
the mutant enzyme has 16% of wild type activity
E763D
-
the mutant enzyme has 1% of wild type activity
E763Q
-
the mutant enzyme has 32% of wild type activity
E816A
-
the mutant enzyme has 76% of wild type activity
F176A/K177A
-
lethal at all temperatures
F44A/L45A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
F556A
-
the mutant has 1% of wild type activity
F556L
-
the mutant has 3% of wild type activity
F609A
-
the mutant enzyme has 117% of wild type activity
F659A
-
the mutant enzyme has 96% of wild type activity
F679A
-
the mutant enzyme has 5% of wild type activity
F679H
-
the mutant enzyme has 13% of wild type activity
F679I
-
the mutant enzyme has 18% of wild type activity
F679L
-
the mutant enzyme has 55% of wild type activity
F679N
-
the mutant enzyme has 18% of wild type activity
F679V
-
the mutant enzyme has 20% of wild type activity
F814A
-
the mutant enzyme has 99% of wild type activity
F815A
-
the mutant enzyme has 92% of wild type activity
G600A
-
in D1 fragment, amino acids 498-844, 4% activity remaining
H260A/S261A
-
modest defect, grows well at 19, 30 and 34°C, but very slowly at 37°C
H682N
-
the mutant enzyme has 1% of wild type activity
H682Q
-
the mutant enzyme has 4% of wild type activity
I47A/S48A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
K111A/R112A
-
modest defect, grows well at 19, 30 and 34°C, but very slowly at 37°C
K156A
-
viable at 19°C and 30°C, but fails to grow at 34°C
K156A/L157A
-
lethal at all temperatures
K223A/D223A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
K573A
-
the mutant enzyme has less than 1% of wild type activity
K573Q
-
the mutant enzyme has less than 1% of wild type activity
K573R
-
the mutant enzyme has 1% of wild type activity
K607A
-
the mutant enzyme has101% of wild type activity
K607Q
-
the mutant enzyme has 74% of wild type activity
K607R
-
the mutant enzyme has 13% of wild type activity
L167A
-
viable at 19°C and 30°C, but fails to grow at 34°C
L16A/P17A
-
temperature-sensitive vD12 allele, forms small colonies at 19°C and fails to grow at 30°C or higher
N120A/N121A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
N126A/N127A
-
modest defect, grows well at 19, 30 and 34°C, but very slowly at 37°C
N42A-Y43A
-
temperature-sensitive vD12 allele, does not grow at 37°C
N54A/R55A
-
modest defect, grows well at 19 and 30°C, but slowly at 34 and very slowly at 37°C
N550A
-
the mutant has 3% of wild type activity
N550D
-
the mutant has 19% of wild type activity
N550Q
-
the mutant has 10% of wild type activity
N570A
-
the mutant enzyme has 37% of wild type activity
N570D
-
the mutant enzyme has 21% of wild type activity
N570Q
-
the mutant enzyme has 73% of wild type activity
N601A
-
the mutant enzyme has 32% of wild type activity
P158A/T159A
-
temperature-sensitive vD12 allele, only grows at 19°C
P35A/S36A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
Q678A
-
the mutant enzyme has 55% of wild type activity
R280A-R281A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
R548A
-
the mutant has 59% of wild type activity
R560A
-
the mutant has 18% of wild type activity
R560K
-
the mutant has 84% of wild type activity
R560Q
-
the mutant has 84% of wild type activity
R562A
-
the mutant has 10% of wild type activity
R562K
-
the mutant has 25% of wild type activity
R562Q
-
the mutant has 23% of wild type activity
R632K
-
the mutant enzyme has 72% of wild type activity
R632Q
-
the mutant enzyme has 58% of wild type activity
R655A
-
the mutant has 31% of wild type activity
R794A
-
the mutant has 124% of wild type activity
R808A
-
the mutant has 142% of wild type activity
S186A/D187A
-
modest defect, grows well at 19, 30 and 34°C, but very slowly at 37°C
S569A
-
the mutant enzyme has 39% of wild type activity
V750A
-
the mutant enzyme has 59% of wild type activity
W189A/L190A
-
temperature-sensitive vD12 allele, does not grow at 37°C
W677A
-
the mutant enzyme has 4% of wild type activity
Y258A-V259A
-
modest defect, grows well at 19, 30 and 34°C, but slowly at 37°C
Y39A/G40A
-
temperature-sensitive vD12 allele, does not grow at 37°C
Y555A
-
the mutant has 2% of wild type activity
Y555F
-
the mutant has 94% of wild type activity
Y555L
-
the mutant has 2% of wild type activity
Y555S
-
the mutant has 4% of wild type activity
Y608A
-
the mutant enzyme has 3% of wild type activity
Y608F
-
the mutant enzyme has 74% of wild type activity
Y608L
-
the mutant enzyme has 62% of wild type activity
Y608S
-
the mutant enzyme has 2% of wild type activity
Y683A
-
in D1 fragment, amino acids 498-844, 0.05% activity remaining, unable to crosslink the cap in presence of S-adenosyl-L-homocysteine
Y683F
-
in D1 fragment, amino acids 498-844, 4% activity remaining
Y683S
-
in D1 fragment, amino acids 498-844, catalytically defective, unable to crosslink the cap in presence of S-adenosyl-L-homocysteine
Y764A
-
the mutant enzyme has 18% of wild type activity
Y836A
-
the mutant enzyme has 15% of wild type activity
E1764A
-
mutation abolishes both guanine-N-7 (G-N-7) and ribose 2'-O methylation methylation
F1691A
-
mutation abolishes both guanine-N-7 (G-N-7) and ribose 2'-O methylation. Alanine substitution results in recombinant virus that does not form plaques and significant diminished viral replication
F1691W
-
mutation does not significantly affect the size of viral plaques. Mutant replicates as wild-type VSV. Mutant exhibits 82% of wild-type guanine-N-7 (G-N-7) methylation
F1691Y
-
mutation does not significantly affect the size of viral plaques. Mutant replicates as wild-type VSV. Mutant exhibits 85% of wild-type guanine-N-7 (G-N-7) methylation
F1745A
-
mutant shows a moderate defect in guanine-N-7 (G-N-7) methylation
F1816A
-
mutant does not show a significant defect in guanine-N-7 (G-N-7) methylation
L1757A
-
mutant does not show a significant defect in guanine-N-7 (G-N-7) methylation
N1692A
-
mutant shows a moderate defect in guanine-N-7 (G-N-7) methylation
P1709A
-
mutant does not show a significant defect in guanine-N-7 (G-N-7) methylation
S1693A
-
mutant shows a moderate defect in guanine-N-7 (G-N-7) methylation
S1827A
-
mutant shows a moderate defect in guanine-N-7 (G-N-7) methylation
W1744A
-
mutant does not show a significant defect in guanine-N-7 (G-N-7) methylation
Y1650A
-
mutation abolishes both guanine-N-7 (G-N-7) and ribose 2'-O methylation. Alanine substitution results in recombinant virus that does not form plaques and significant diminished viral replication
Y1650F
-
mutation does not significantly affect the size of viral plaques. Mutant replicates as wild-type VSV. Mutant exhibits 88% of wild-type guanine-N-7 (G-N-7) methylation
Y1650W
-
mutation does not significantly affect the size of viral plaques. Mutant replicates as wild-type VSV. Mutant exhibits 90% of wild-type guanine-N-7 (G-N-7) methylation
Y1835A
-
mutant does not show a significant defect in guanine-N-7 (G-N-7) methylation
E149A
-
the mutant shows about 1% N7 MTase activity as compared to the wild type protein
E34A
-
the mutant shows about 70% N7 MTase activity as compared to the wild type protein
F133A
-
the mutant shows about 99% N7 MTase activity as compared to the wild type protein
H42A
-
the mutant shows wild type N7 MTase activity
K21A
-
the mutant shows about 95% N7 MTase activity as compared to the wild type protein
K41A
-
the mutant shows about 90% N7 MTase activity as compared to the wild type protein
K45A
-
the mutant shows about 105% N7 MTase activity as compared to the wild type protein
L16A
-
the mutant shows about 110% N7 MTase activity as compared to the wild type protein
L184A
-
the mutant shows about 85% N7 MTase activity as compared to the wild type protein
Q114A
-
the mutant shows about 97% N7 MTase activity as compared to the wild type protein
R213A
-
the mutant shows about 30% N7 MTase activity as compared to the wild type protein
R37A
-
the mutant shows about 5% N7 MTase activity as compared to the wild type protein
R44A
-
the mutant shows about 65% N7 MTase activity as compared to the wild type protein
R57A
-
the mutant shows about 10% N7 MTase activity as compared to the wild type protein
R84A
-
the mutant shows about 3% N7 MTase activity as compared to the wild type protein
T216A
-
the mutant shows wild type N7 MTase activity
V55A
-
the mutant shows about 50% N7 MTase activity as compared to the wild type protein
W87A
-
the mutant shows about 10% N7 MTase activity as compared to the wild type protein
Y220A
-
the mutant shows about 30% N7 MTase activity as compared to the wild type protein
Y254A
-
the mutant shows about 110% N7 MTase activity as compared to the wild type protein
D70A
lethal
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
D70E
growth at all temperatures
D70N
lethal
D78A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 30°C and 37°C, but at 25°C
D78A
lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
D78E
growth at all temperatures except at 37°C
D78N
growth at all temperatures
D94A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
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
D94E
growth at all temperatures
D94N
lethal
E225A
growth at all temperatures except at 37°C
F141A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
F141A
suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
F214A
growth at all temperatures except at 37°C
H144A
growth at all temperatures
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
K267A
growth at all temperatures
K54A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, inactive mutant in vivo
K54A
lethal, suppresses methyltransferase activity to less than 3% of the wild-type level, in vivo lethality correlates with loss of catalytic activity
K54Q
lethal
K54R
lethal
K75A
-
site-directed mutagenesis, some complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype
K81A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant, mutation is lethal in vivo
K81Q
lethal
K81R
lethal
N51A
-
site-directed mutagenesis, no complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C
N51D
lethal
R106A
-
site-directed mutagenesis, only slight complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C
R106K
lethal
R106Q
lethal
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
R59A
growth at all temperatures
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
R84A
growth at all temperatures
Y124A
-
site-directed mutagenesis, complementation of the yeast abd1DELTA mutant at 25°C and 30°C, not at 37°C, temperature-sensitive phenotype
Y145A
lethal
Y145F
grows as well as the wild-type ECM1 strain at all temperatures
Y145F
growth at all temperatures
Y145L
grows as well as the wild-type ECM1 strain at all temperatures
Y145L
growth at all temperatures
Y145S
lethal
Y212A
growth at all temperatures except at 37°C
C382Y
15-50% of wild-type activity
C382Y
72% of wild-type methylation activity
C414R
complete loss of methylation activity
C414R
mutation abolishes the N7-MTase activity on GTP
D243A
-
the point mutation in the ExoN domain does not significantly interfere with the N7-MTase function
D243A
79% of wild-type methylation activity
D331A
-
the mutation destroys the MTase activity of nsp14 in vivo
D331A
complete loss of methylation activity
D331A
mutation abolishes the N7-MTase activity on GTP
D331Y
complete loss of methylation activity
D331Y
mutation abolishes the N7-MTase activity on GTP
D352A
15-50% of wild-type activity
D352A
47% of wild-type methylation activity
D90A/E92A
-
the point mutation in the ExoN domain does not significantly interfere with the N7-MTase function
D90A/E92A
93% of wild-type methylation activity
F73A
91% of wild-type methylation activity
F73A
mutation abolishes the N7-MTase activity on GTP
G333A
complete loss of methylation activity
G333A
mutation abolishes the N7-MTase activity on GTP
H268L
-
the point mutation in the ExoN domain does not significantly interfere with the N7-MTase function
H268L
85% of wild-type methylation activity
I332A
15-50% of wild-type activity
I332A
83% of wild-type methylation activity
K336A
15-50% of wild-type activity
K336A
50% of wild-type methylation activity
P335A
61% of wild-type methylation activity
P335A
mutation abolishes the N7-MTase activity on GTP
R310A
complete loss of methylation activity
R310A
mutation abolishes the N7-MTase activity on GTP
R84A
6% of wild-type methylation activity
R84A
mutation abolishes the N7-MTase activity on GTP
T428A
15-50% of wild-type activity
T428A
27% of wild-type methylation activity
W86A
complete loss of methylation activity
W86A
mutation abolishes the N7-MTase activity on GTP
Y368A
complete loss of methylation activity
Y368A
mutation abolishes the N7-MTase activity on GTP
Y420A
15-50% of wild-type activity
Y420A
5% of wild-type methylation activity
F141A/R142A
-
grows slowly at 19 and 30°C and fails to grow at 34°C or higher
F141A/R142A
-
temperature-sensitive vD12 allele, does not grow above 34°C
H682A
-
in D1 fragment, amino acids 498-844, 4% activity remaining, unable to crosslink the cap in presence of S-adenosyl-L-homocysteine
H682A
-
the mutant enzyme has less than 1% of wild type activity
additional information
-
mapping of lipid-binding site, membrane binding activity of wild-type and diverse constructed mutants
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
additional information
-
construction of knock-down HeLa cells S3, gene inactivation by small interference RNA in HeLa cells results in apoptosis
additional information
-
exchange of 8 residues for alanine in the D1 fragment, amino acids 498-844
additional information
-
exchange for alanine at single sites within the methyltransferase domain fragment of D1, loss of activity when exchanged hits His682 and Tyr683, active site
additional information
-
construction of carboxyl-truncated D1 genes, amino acid 1-666 and 1-425: 347 residues are sufficient for activity