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Y110F
no significant change in products
Y110L
remodeling the active site to alter the electronics results in delayed timing of the hydride migration from methyl attack of the DELTA24-bond, that thereby produces metabolic switching of product ratios in favor of DELTA25(27)-olefins or impairs the second C1-transfer activity
C128S
-
Km (mM) (substrate: AdoMet): 30, kcat (1/sec) (substrate: AdoMet): 0.005, Kd (mM) (zymosterol): 0.005, Kd (mM) (AdoMet): 0.008
D229L
relative activity: 100%
E224L
relative activity: 74%
F183L
100% relative enzyme activity
H224L
-
the ratio of turnover-number to KM-value for zymosterol is 1.4fold lower than the wild-type ratio
K353L
56% relative enzyme activity
S87E
100% relative enzyme activity
S87Q
100% relative enzyme activity
S97E
relative activity: 100%
S97Q
relative activity: 100%
T219L
32% relative enzyme activity, mutant can perform C2-transfer activity to give 24-ethyl(idene)-sterols
V222L
27% relative enzyme activity
Y192F
100% relative enzyme activity, mutant can perform C2-transfer activity to give 24-ethyl(idene)-sterols
Y192L
95% relative enzyme activity
Y81A
-
Km (mM): 0.018 (zymosterol), 0.023 (26,27-dehydrozymosterol), kcat (1/sec): 0.00083 (zymosterol), 0.000116 (26,27-dehydrozymosterol), Ki (mM): 0.011 (26,27-dehydrozymosterol), 0.00019 (25-azalanosterol), 0.00013 (24(R,S),25-epiminolanosterol), 0.071 (ergosterol)
Y81I
-
Km (mM): 0.021 (zymosterol), 0.027 (26,27-dehydrozymosterol), kcat (1/sec): 0.00433 (zymosterol), 0.00025 (26,27-dehydrozymosterol), Ki (mM): 0.009 (26,27-dehydrozymosterol), 0.00020 (25-azalanosterol), 0.00016 (24(R,S),25-epiminolanosterol), 0.077 (ergosterol)
Y81V
-
Km (mM): 0.026 (zymosterol), 0.024 (26,27-dehydrozymosterol), kcat (1/sec): 0.00116 (zymosterol), 0.00011 (26,27-dehydrozymosterol), Ki (mM): 0.007 (26,27-dehydrozymosterol), 0.000195 (25-azalanosterol), 0.000145 (24(R,S),25-epiminolanosterol), 0.074 (ergosterol)
Y81W
-
Km (mM): 0.017 (zymosterol), 0.023 (26,27-dehydrozymosterol), kcat (1/sec): 0.0133 (zymosterol), 0.0033 (26,27-dehydrozymosterol), Ki (mM): 0.010 (26,27-dehydrozymosterol), 0.00039 (25-azalanosterol), 0.00036 (24(R,S),25-epiminolanosterol), 0.088 (ergosterol)
Y177F
-
mutant proteins catalyses zymosterol much less efficiently than the wild-type enzyme, with a Vmax/Km ratio 23-40% that of wild-type
Y208F
-
mutant proteins catalyses zymosterol much less efficiently than the wild-type enzyme, with a Vmax/Km ratio 23-40% that of wild-type
Y66F
-
mutant proteins catalyses zymosterol much less efficiently than the wild-type enzyme, with a Vmax/Km ratio 23-40% that of wild-type. Sterol composition of the Y66F catalysis is different. The sterol mixture possesses a significant decrease in ergosta-8,24(25)-dienol compensated by an increase in ergosta-8,25(27)-dienol and ergosta-8,24(28)-dienol
A193L
no activity
A193S
95% relative enzyme activity
A193S
relative activity: 95%
A196L
73% relative enzyme activity
A196L
relative activity: 73%
A200L
4% relative enzyme activity
A200L
relative activity: 4%
A221L
25% relative enzyme activity
A221L
relative activity: 25%
A350L
100% relative enzyme activity
A350L
relative activity: 100%
C128L
no activity
C128L
-
no activity with AdoMet as substrate, Kd (mM) (zymosterol): 0.005, Kd (mM) (AdoMet): 0.028, photolabelled: value below 1%
C198L
95% relative enzyme activity
C198L
relative activity: 95%
C198V
16% relative enzyme activity
C198V
relative activity: 16%
D125L
no activity
D125L
-
no activity with AdoMet as substrate, Kd (mM) (zymosterol): 0.006, Kd (mM) (AdoMet): 0.012, photolabelled: 30%
D152L
no activity
D152L
-
no activity with AdoMet as substrate, Kd (mM) (zymosterol): 0.005, Kd (mM) (AdoMet): 0.014, photolabelled: 35%
D189L
-
the ratio of turnover-number to KM-value for zymosterol is 1.4fold lower than the wild-type ratio
D189L
relative activity: 91%
D276L
no activity
D65L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, unchanged activity
D65L
relative activity: 100%
D79L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, not decreased activity, 1 new activity to form 24-methylzymosterol from zymosterol
D79L
relative activity: 81%
E108L
-
the ratio of turnover-number to KM-value for zymosterol is 11fold lower than the wild-type ratio
E108L
relative activity: 34%
E195L
no activity
E209L
-
the ratio of turnover-number to KM-value for zymosterol is 2.4fold lower than the wild-type ratio
E209L
relative activity: 79%
E246L
-
the ratio of turnover-number to KM-value for zymosterol is 13.5fold lower than the wild-type ratio
E246L
relative activity: 6%
E64L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, unchanged activity
E64L
relative activity: 100%
E68L
no activity
E68L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, no activity
E82D
55% relative enzyme activity
E82D
relative activity: 55%
E82L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, not decreased activity, 1 new activity to form 24-methylzymosterol from zymosterol
E82L
-
produces plant-like substrate-product profiles
E82L
relative activity: 99%
E82Q
90% relative enzyme activity
E82Q
relative activity: 90%
E98L
-
potentially responsible for substrate binding to leucine by site directed mutagenesis, unchanged activity
E98L
relative activity: 100%
F178L
34% relative enzyme activity
F178L
relative activity: 34%
F188L
2% relative enzyme activity
F188L
relative activity: 2%
F220L
29% relative enzyme activity
F220L
relative activity: 29%
F269L
no activity
F357L
100% relative enzyme activity
F357L
relative activity: 100%
F89L
100% relative enzyme activity
F89L
relative activity: 100%
F91L
15% relative enzyme activity
F91L
relative activity: 15%
G127L
1% relative enzyme activity
G127L
relative activity: 1%
G129L
no activity
G131L
5% relative enzyme activity
G131L
relative activity: 5%
G132L
50% relative enzyme activity
G132L
relative activity: 50%
G217L
100% relative enzyme activity, mutant can perform C2-transfer activity to give 24-ethyl(idene)-sterols
G217L
relative activity: 100%
G218L
100% relative enzyme activity, mutant can perform C2-transfer activity to give 24-ethyl(idene)-sterols
G218L
relative activity: 100%
G347L
5% relative enzyme activity
G347L
relative activity: 5%
G351L
90% relative enzyme activity
G351L
relative activity: 90%
G352L
3% relative enzyme activity
G352L
relative activity: 3%
G84L
14% relative enzyme activity
G84L
relative activity: 14%
G86L
16% relative enzyme activity
G86L
relative activity: 16%
H107L
-
the ratio of turnover-number to KM-value for zymosterol is 2.4fold lower than the wild-type ratio
H107L
relative activity: 23%
H199L
-
the ratio of turnover-number to KM-value for zymosterol is 162fold lower than the wild-type ratio
H199L
relative activity: 2%
H199Q
45% relative enzyme activity
H199Q
relative activity: 45%
H199R
20% relative enzyme activity
H199R
relative activity: 20%
H238L
-
the ratio of turnover-number to KM-value for zymosterol is 1.6fold lower than the wild-type ratio
H238L
relative activity: 85%
H90L
no activity
H90Q
no activity
H90R
no activity
I194L
67% relative enzyme activity
I194L
relative activity: 67%
K215L
100% relative enzyme activity
K215L
relative activity: 100%
P133L
no activity
P133L
-
no activity with AdoMet as substrate, Kd (mM) (zymosterol): 0.015, Kd (mM) (AdoMet): no binding, photolabelled: value below 1%
P201L
58% relative enzyme activity
P201L
relative activity: 58%
P216L
65% relative enzyme activity
P216L
relative activity: 65%
S354L
100% relative enzyme activity
S354L
relative activity: 100%
S87L
100% relative enzyme activity
S87L
relative activity: 100%
S88L
61% relative enzyme activity
S88L
relative activity: 61%
S88N
17% relative enzyme activity
S88N
relative activity: 17%
T197L
93% relative enzyme activity
T197L
relative activity: 93%
V126L
1% relative enzyme activity
V126L
relative activity: 1%
V130L
100% relative enzyme activity
V130L
relative activity: 100%
V349L
47% relative enzyme activity
V349L
relative activity: 47%
W225F
100% relative enzyme activity
W225F
relative activity: 60%
W225L
100% relative enzyme activity
W225L
relative activity: 100%
W286F
100% relative enzyme activity
W286F
relative activity: 51%
W286L
2% relative enzyme activity
W286L
relative activity: 2%
W85F
100% relative enzyme activity
W85F
relative activity: 55%
W85L
40% relative enzyme activity
W85L
relative activity: 40%
Y153F
100% relative enzyme activity
Y153F
-
Km (mM) (substrate: AdoMet): 0.016, kcat (1/sec) (substrate: AdoMet): 0.003, Kd (mM) (zymosterol): 0.007, Kd (mM) (AdoMet): 0.013, photolabelled: 55%
Y153F
relative activity: 41%
Y153L
no enzyme activity
Y153L
-
Km (mM) (substrate: AdoMet): 0.017, kcat (1/sec) (substrate: AdoMet): 0.01, Kd (mM) (zymosterol): 0.004, Kd (mM) (AdoMet): 0.004
Y153L
relative activity: 100%
Y207F
100% relative enzyme activity
Y207F
relative activity: 100%
Y207L
3% relative enzyme activity
Y207L
relative activity: 3%
Y223F
100% relative enzyme activity, mutant can perform C2-transfer activity to give 24-ethyl(idene)-sterols
Y223F
relative activity: 100%
Y223L
100% relative enzyme activity
Y223L
relative activity: 100%
Y287L
20% relative enzyme activity
Y287L
relative activity: 20%
Y74F
100% relative enzyme activity
Y74F
relative activity: 100%
Y74L
100% relative enzyme activity
Y74L
relative activity: 100%
Y81F
-
produces plant-like substrate-product profiles
Y81F
-
Km (mM): 0.017 (zymosterol), 0.026 (26,27-dehydrozymosterol), kcat (1/sec): 0.0108 (zymosterol), 0.0025 (26,27-dehydrozymosterol), Ki (mM): 0.010 (26,27-dehydrozymosterol), 0.00036 (25-azalanosterol), 0.00031 (24(R,S),25-epiminolanosterol), 0.082 (ergosterol)
Y81F
relative activity: 100%
Y81L
90% relative enzyme activity
Y81L
-
Km (mM): 0.018 (zymosterol), 0.024 (26,27-dehydrozymosterol), kcat (1/sec): 0.00416 (zymosterol), 0.00033 (26,27-dehydrozymosterol), Ki (mM): 0.011 (26,27-dehydrozymosterol), 0.00021 (25-azalanosterol), 0.00018 (24(R,S),25-epiminolanosterol), 0.078 (ergosterol)
Y81L
relative activity: 90%
Y83F
95% relative enzyme activity
Y83F
relative activity: 95%
Y83L
90% relative enzyme activity
Y83L
relative activity: 90%
Y83W
100% relative enzyme activity
Y83W
relative activity: 100%
additional information
-
natural amphotericine B-resistant mutant strain
additional information
-
C57BL/6 mice immunized with a vaccine candidate consisting of 24-c-methyltransferase as an antigene formulated monophosphoryl lipid A (MPL-SE) show Ag-specific Th1 immune responses characterized by robust production of IFN-gamma upon specific Ag re-exposure in vitro. Upon challenge with Leishmania infantum, mice immunized with SMT plus MPL-SE show significant lower parasite burdens in both spleens and livers compared with non-immunized mice or mice injected with adjuvant alone
additional information
C57BL/6 mice immunized with a vaccine candidate consisting of 24-c-methyltransferase as an antigene formulated monophosphoryl lipid A (MPL-SE) show Ag-specific Th1 immune responses characterized by robust production of IFN-gamma upon specific Ag re-exposure in vitro. Upon challenge with Leishmania infantum, mice immunized with SMT plus MPL-SE show significant lower parasite burdens in both spleens and livers compared with non-immunized mice or mice injected with adjuvant alone
additional information
-
Leishmania major promastigotes cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22,26-azasterol
additional information
-
by photoaffinity labeling and mutational analysis the AdoMet binding site is defined. Results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet
additional information
-
scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicate that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C1-transfer activity to include plant-like C2-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Saccharomyces cerevisiae Erg6p regulate the course of the C-methylation reaction toward product differences
additional information
scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicate that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C1-transfer activity to include plant-like C2-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Saccharomyces cerevisiae Erg6p regulate the course of the C-methylation reaction toward product differences