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A71V
mutation confers resistance to antimalarial DHFR inhibitor drug cycloguanil, mutation is in the DHFR domain, close to the binding sites for DHF
G137D
mutation confers resistance to antimalarial DHFR inhibitor drugs pyrimethamine and cycloguanil, mutation is in the DHFR domain, close to the binding sites for DHF
F96I
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
V77A/I130M/I138V
site-directed mutagenesis
Y102F
the mutant shows reduced catalytic efficiency compared to the wild type enzyme. The affinities of the antifolates increase up to 60fold with this mutant
I26N/K31P
less than 10% of wild-type activity
K31P
no detectable activity
L22R
mutation predicted in silico to be resistant to methotrexate, confers resistance to methotrexate to transfected CHO cells. 65% of wild-type enzyme activity
L30Q
mutation found in a methotrexate resistant cell line, confers resistance to methotrexate to transfected CHO cells. 42% of wild-type enzyme activity
Q134K
51% of wild-type enzyme activity
C85A/C152S
kinetic properties similar to wild-type
C85S/C152E
-
cysteine-free mutant, analysis of inhibition by p.eroxynitrite. bicarbonate buffer protects mutant from inhibition by peroxynitrite. Decrease in mutant stability upon oxidation
D27S
site-directed mutagenesis, the mutant shows a 3400fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type
DELTAAla145
the mutant shows increased catalytic efficiency compared to the wild type enzyme
DELTAArg52
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAArg52/DELTAAla145
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAArg52/DELTAGly121
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAArg52/DELTAGly67
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAGly121
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAGly121/DELTAAla145
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAGly67
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAGly67/DELTAAla145
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
DELTAGly67/DELTAGly121
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
I14A
the mutant shows strongly reduced pre-steady-state rates of H transfer at 25°C and pH 7.0 compared to the wild type enzyme
I14G
the mutant shows severely reduced pre-steady-state rates of H transfer at 25°C and pH 7.0 compared to the wild type enzyme
I14V
the mutant shows reduced pre-steady-state rates of H transfer at 25°C and pH 7.0 compared to the wild type enzyme
I68M
compared to wild-type, low kcat/Km (DHF) values. Mutant allows growth in presence of sorbitol up to 1.44 osmol conditions
K32M
-
weakens binding of dihydrofolate over 60-fold, increases kcat value by a factor of 5
L16M
-
site-directed mutagenesis, same kinetic properties like the wild-type enzyme
L16M/L20M
-
site-directed mutagenesis, double mutant, elevated turnover number and specific activity
L16M/L20M/L42M
-
site-directed mutagenesis, triple mutant, elevated turnover number and specific activity
L16SeM
-
site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme
L16SeM/L20SeM
-
site-directed mutagenesis, double mutant, with selenomethionine, elevated turnover number and specific activity
L16SeM/L20SeM/L42SeM
-
site-directed mutagenesis, triple mutant, with selenomethionine, elevated turnover number and specific activity
L20M
-
site-directed mutagenesis, elevated turnover number and specific activity
L20SeM
-
site-directed mutagenesis, with selenomethionine, elevated turnover number and specific activity
L28F
mutant behaves similarly to wild-type
L42M
-
site-directed mutagenesis, same kinetic properties like the wild-type enzyme
L42SeM
-
site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme
L54I
-
the mutation reduces the hydride transfer efficiency by about 100fold
L92M
-
site-directed mutagenesis, same kinetic properties like the wild-type enzyme
L92SeM
-
site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme
M1A/C85A/C152S
kinetic properties similar to wild-type
M1A/M16F/M20L/M42Y/M92F/C85A/C152S
hyperactive mutant, increase in dissociation rate constant of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex
M1A/M16N/M20L/M42Y/C85A/M92F/C152S
mutant ANLYF carries seven amino acid substitutions that result in a methionine- and cysteine-free mutant enzyme with decreased stability. Mutant ANLYF show very high activity and may be stabilized by backbone cyclization via a cyanocysteine-mediated intramolecular ligation reaction, without loss of its high activity
M1A/M16N/M20L/M42Y/M92F/C85A/C152S
hyperactive mutant, increase in dissociation rate constant of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex
M1A/M16S/M20L/M42Y/M92F/C85A/C152S
hyperactive mutant, increase in dissociation rate constant of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex
M1P/C85A/C152S
kinetic properties similar to wild-type
M1S/C85A/C152S
kinetic properties similar to wild-type
M42A
-
45% decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42C
-
slight decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42E
-
6fold decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42G
-
45% decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42H
-
40% decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42I
-
slight increase in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42L
-
slight decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42P
-
50% increase in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42Q
-
45% decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42S
-
45% decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42T
-
slight increase in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42V
-
slight decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42W/G121V
-
kinetic isotope effect study, major change in the nature of H transfer, leading to poor reorganization and substantial gating
M42Y
-
30% increase in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
R44A
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44C
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44D
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44E
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44F
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44G
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44I
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44K
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44L
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44M
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44N
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44P
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44Q
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44S
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44T
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44V
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44W
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
R44Y
-
the mutation significantly reduces the enzymatic activity and the binding affinity toward the cofactor NADPH
Y100F
site-directed mutagenesis, the mutant shows a 14fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type
Y100F/D27S
site-directed mutagenesis, the mutant shows an over 100000fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type
Y69L
compared to wild-type, low kcat/Km (DHF) values. Mutant allows growth in presence of sorbitol up to 0.81 osmol conditions
A31K
-
site-directed mutagenesis
L30K
-
site-directed mutagenesis
L30K/A31K
-
site-directed mutagenesis
C829T
-
naturally occurring single nucleotide polymorhism , near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase leads to a decrease in microRNA binding, which in turn leads to overexpression of its target and results in resistance to methotrexate
F31A/F34V/Q35H
methotrexate-resistant mutant, more than 4000fold decrease in ratio kcat/KM
F31G/Q35N
methotrexate-resistant mutant, more than 50fold decrease in ratio kcat/KM
F31P
methotrexate-resistant mutant, more than 30fold decrease in ratio kcat/KM
F31P/Q35E
methotrexate-resistant mutant, more than 200fold decrease in ratio kcat/KM
F31P/Q35H
methotrexate-resistant mutant, more than 150fold decrease in ratio kcat/KM
F31R/F34A/Q35N
methotrexate-resistant mutant, more than 5000fold decrease in ratio kcat/KM
F31R/F34T/Q35R
methotrexate-resistant mutant, more than 7000fold decrease in ratio kcat/KM
F31R/F34T/Q35S
methotrexate-resistant mutant, more than 2000fold decrease in ratio kcat/KM
F31R/Q35E
methotrexate-resistant mutant, more than 70fold decrease in ratio kcat/KM
F31S/Q35E
methotrexate-resistant mutant, more than 450fold decrease in ratio kcat/KM
Q35K/N64S
site-directed mutagenesis
Q35S/N64F
site-directed mutagenesis
V115A
-
mutant resistant to methotrexate, 600-fold increase in Ki value compared with wild-type
V115C
-
mutant resistant to methotrexate, 600-fold increase in Ki value compared with wild-type
L62F
thermosensitive, mutant strain is to grow at 30°C but not at 40°C. Mutant protein shows a reduction in DHFR activity from 1.51 to 0.06 Units/mg protein, resulting in increasing DHF amount at the expense of THF. Total folate production by the mutant in two-stage fermentation process with temperature shift-up from 30°C to 40°C increases by three-fold compared with the parental strain
R53M
-
resistance providing naturally occurring mutant from methotrexate-resistant strain, lower turnover number
D31A
7% of wild-type activity
D31E
18.9% of wild-type activity
D31L
0.39% of wild-type activity
D31N
15.4% of wild-type activity
D31Q
14.7% of wild-type activity
L32A
96% of wild-type activity. 12-fold decrease in affinity for trimethoprim
L32D
33% of wild-type activity. 7-fold decrease in affinity for trimethoprim
L32F
103% of wild-type activity
V76A
98.7% of wild-type activity
A16V
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
A16V/N51I/C59R/S108N
-
point mutations of the active site residues lead to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine, binding structure modelling, overview
C59R
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
I164L
-
mutation I164L is not associated with high-level sulfadoxine-pyrimethamine resistance or poor outcome among infected adults living where malaria is highly endemic
I164X
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
I51N/C59R/N108S/I164L
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
K27E
kinetic properties similar to wild-type, increase in solubility
N51I
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
N51I/C59R/N108S/I164L
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
N51I/C59R/N108T/I164L
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
N51I/C59R/S108N/I164L/D187A
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
N51I/C59R/S108N/I164L/I150V/N182I/N201D
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
N51I/C59R/S108N/I164L/K96N
-
mutant isolated in an effort to mimic the impact of drug pressure on the selection of dihydrofolate reductase mutations in the malaria parasite population, based on DHFR-TS-QM mutation which exhibits the highest degree of antifolate resistance developed in the field
N5I/C59R/S108N/I164L
naturally occuring mutant. Residue N108 is the cause of pyrimethamine resistance with the highest repulsive interaction energy
S108A
-
combinatorial mutagenesis
S108C
-
combinatorial mutagenesis
S108D
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108E
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108F
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108G
-
combinatorial mutagenesis
S108H
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108I
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108L
-
combinatorial mutagenesis, reduced activity
S108M
-
combinatorial mutagenesis, reduced activity
S108N/C59R
-
natural mutants isolated in a study on the association between the clinical and parasitological response to sulfadoxinepyrimethamine and allelic combinations of dihydrofolate reductase and dihydropteroate synthase genes
S108N/N51I
-
natural mutants isolated in a study on the association between the clinical and parasitological response to sulfadoxinepyrimethamine and allelic combinations of dihydrofolate reductase and dihydropteroate synthase genes
S108P
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108Q
-
combinatorial mutagenesis, reduced activity, conferred resistance to pyrimethamine and cycloguanil
S108R
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108V
-
combinatorial mutagenesis, reduced activity
S108W
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
S108Y
-
combinatorial mutagenesis, no detectable activity on refolding from inclusion bodies
W48Y
-
3% of wild-type activity
W48Y/N188Y
-
28.9% of wild-type activity
Y35G/F37L
kinetic properties similar to wild-type, increase in solubility
Y35L/F37R
kinetic properties similar to wild-type, increase in solubility
Y35L/F37T
kinetic properties similar to wild-type, increase in solubility
Y35Q/F37R
kinetic properties similar to wild-type, increase in solubility
F69N
site-directed mutagenesis
F69S
site-directed mutagenesis
K37Q
site-directed mutagenesis, the mutant exhibits increased sensitivity for inhibition by 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine compared to the wild-type enzyme
K37Q/F69N
site-directed mutagenesis, the mutant exhibits increased sensitivity for inhibition by 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine compared to the wild-type enzyme. Structure analysis of the mutant in complex with TMP, overview
K37Q/F69S
site-directed mutagenesis
K37S
site-directed mutagenesis
K37S/F69N
site-directed mutagenesis
K37S/F69S
site-directed mutagenesis
F69F
site-directed mutagenesis
F69N
site-directed mutagenesis
S37K
site-directed mutagenesis
S37K/S69F
site-directed mutagenesis
S37K/S69N
site-directed mutagenesis
S37Q/S69F
site-directed mutagenesis
S37Q/S69N
site-directed mutagenesis
S73Q
site-directed mutagenesis
F98Y
site-directed mutagenesis, the mutant enzyme shows reduced sensitivity to inhibitors compared to the wild-type enzyme, structure comparison to the wild-type, overview
N48E/N130D
site-directed mutagenesis, the S1 mutant enzyme shows improved expression levels and solubility. Inhibition kinetics and inhibitor binding thermodynamics in comparison to the wild-type enzyme, overview. In the absence of substrate and cofactor the active site of S1 DHFR is blocked, trimethoprim shows loss of potency and NADPH synergy on binding S1 DHFR
N48E/N130D/Y98F/A43G
site-directed mutagenesis, inhibition kinetics and inhibitor binding thermodynamics in comparison to the wild-type enzyme, overview
Y98F/A43G
inhibitor trimethoprim shows loss potency and NADPH synergy on binding S1 mutant DHFR. Mutation of residues Y98F/A43G in S1 mutant restores trimethoprim sensitivity and NADPH synergy
H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T
Km or kcat (NADPH or dihydrofolate) much lower compared to wild-type, no difference in NADPH binding compared to wild-type, kcat of mutant is pH dependent (maximal rate at pH 7.0 similar to wild-type), inhibitor trimethoprim is bound tighter to mutant compared to wild-type
H33F
mutant shows a pH profile similar to DHFR from Escherichia coli, kcat values are pH independent over pH 6.0-7.5
V11D
the single amino acid replacement is sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric mutant enzyme V11D is 15 kJ/mol lower than that of the wild-type dimer. Both the steady-state turnover numbers and rates of hydride transfer are reduced in V11D
V126E
mutant enzyme remains as a dimer, steady-state turnover numbers and rates of hydride transfer are reduced
V11D
-
the single amino acid replacement is sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric mutant enzyme V11D is 15 kJ/mol lower than that of the wild-type dimer. Both the steady-state turnover numbers and rates of hydride transfer are reduced in V11D
-
V126E
-
mutant enzyme remains as a dimer, steady-state turnover numbers and rates of hydride transfer are reduced
-
P292A
site-directed mutagenesis, the mutation, reduces the DHFR catalytic efficiency by 7fold
W296A
site-directed mutagenesis, reduces the DHFR catalytic efficiency by 100fold
T86N
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T86S
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
G121V
reduced catalytic activity
G121V
the mutation interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway
G121V
-
kinetic isotope effect study, environmentally coupled tunnelling similar to wild-type
G121V
reduced catalytic efficiency due to significantly reduced pre-exponential factors, the chemical mechanism of catalysis is not impaired
G121V
-
the mutation reduces the hydride transfer efficiency by about 100fold
M42W
-
6fold decrease in ratio kcat/Km, analysis of thermodynamic parameters for urea denaturation
M42W
-
kinetic isotope effect study, environmentally coupled tunnelling similar to wild-type
Q67H
-
about 100-fold tightening in binding to both dihydrolfolate and NADPH, mutation can help rescue the K32 M mutation
Q67H
mutant with reasonable catalytic efficiency, but displays substrate and cofactor inhibition. Contrary to wild-type which allows growth on all sorbitol conditions until the osmolyte concentration becomes too high or cell water content becomes too low, mutant Q67H allows growth up to 1.81 osmol conditions
N64F
site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview
N64F
site-directed mutagenesis, the mutant exhibits increased sensitivity for inhibition by 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine compared to the wild-type enzyme
N64S
site-directed mutagenesis
N64S
site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview
Q35K
site-directed mutagenesis
Q35K
site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview
Q35K/N64F
site-directed mutagenesis
Q35K/N64F
site-directed mutagenesis, active site mutant analogues to the enzyme from Pneumocystis carinii, shows enhanced activity compared to the wild-type enzymekinetic analysis, overview
Q35S
site-directed mutagenesis
Q35S
site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview
Q35S/N64S
site-directed mutagenesis
Q35S/N64S
site-directed mutagenesis, active site mutant analogues to the enzyme from Pneumocystis jirovecii, shows enhanced activity compared to the wild-type enzyme, kinetic analysis, overview
C59R/S108N
-
mutant with increased resistance to anitfolate antimalarials, use for screening inhibitors
C59R/S108N
-
point mutations of the active site residues lead to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine, binding structure modelling, overview
N51I/C59R/S108N
-
natural mutants isolated in a study on the association between the clinical and parasitological response to sulfadoxinepyrimethamine and allelic combinations of dihydrofolate reductase and dihydropteroate synthase genes. The majority of clinical isolates is characterized as quadruple, i.e. 196 isolates with mutations N51I-C59R-S108N in dihydrofolate reductase and A437G dihydropteroate synthase
N51I/C59R/S108N
-
pyrimethamine-resistant mutant, 50% decrease in Km value
N51I/C59R/S108N/I164L
-
mutant with increased resistance to anitfolate antimalarials, use for screening inhibitors
N51I/C59R/S108N/I164L
-
mutant highly resistant to pyrimethamine, but sensitive to WR99210, i.e. DHFT-QM mutant
N51I/C59R/S108N/I164L
-
pyrimethamine-resistant mutant, 50% decrease in Km value
S108N
-
combinatorial mutagenesis, conferred resistance to pyrimethamine and cycloguanil
S108N
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
S108T
-
combinatorial mutagenesis, reduced activity
S108T
-
point mutation of the active site residue leads to a widespread resistance of the parasite to the drugs cycloguanil and pyrimethamine
S58R/S117N
-
double mutant with naturally occurring point mutations, antifolate drug resistant
S58R/S117N
pyrimethamine-resistant mutant
additional information
-
overview about spontaneously occuring mutants and possible mutagenesis studies
additional information
-
transformation of murine dihydrofolate reductase cDNA by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR, which differs from the murine sequence by 19 amino acids. Expression of the bovine dihydrofolate reductase cDNA in bacterial cells produces a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein
additional information
-
use of the dihydrofolate reductase-targeted short hairpin RNA vector for immunoglobulin G expression in geneamplified stable CHO cells. Cells increase IgG expression at 1.0 microM methotrexate by more than 100% and improve the genomic stability of IgG expression in methotrexate-free cultures by approximately 30%
additional information
-
knock-down of dihydrofolate reductase or heart and neural crest derivatives expressed transcript HAND2 in fertilized eggs causes cardiac malformation. Expression of HAND2 is reduced in dihydrofolate reductase knock-down embryos. Microinjection of HAND2 mRNA into fertilized eggs can induce HAND2 overexpression which rescues the cardiac malformation phenotypes of dihydrofolate knock-down embryos
additional information
the C-terminal Strep-tag does not affect enzyme activity or inhibition by RAB-propyl inhibitors
additional information
-
the C-terminal Strep-tag does not affect enzyme activity or inhibition by RAB-propyl inhibitors
additional information
-
the C-terminal Strep-tag does not affect enzyme activity or inhibition by RAB-propyl inhibitors
-
additional information
double-labeling of enzyme with fluorescent quencher QSY35, at amino acid 17, and fluorescent probe Alexa555,at amino acid 35, by introducing Cys residues at these sites. Labeled enzyme retains full catalytic activity, analysis of first order rate constants for the stopped-flow fluorescent change of forward and reverse reaction
additional information
permutations in the N- and C-terminal domain show that for equilibrium and kinetic folding mechanism, a continuous adenosine-binding domain is required for a stable thermal intermediate and a continuous discontinuous loop domain is required for a stable urea intermediate
additional information
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permutations in the N- and C-terminal domain show that for equilibrium and kinetic folding mechanism, a continuous adenosine-binding domain is required for a stable thermal intermediate and a continuous discontinuous loop domain is required for a stable urea intermediate
additional information
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construction of a mutant isoform that carries the K32M mutation in gene copies 1 and 3 and the Q67H mutation in gene copies 2 and 4 of the tetrameric enzyme. Mutation Q67H can mostly compensate for mutation K32M. In a multimutant that combines the K32M mutation in gene copies 1 and 3 with the Q67H mutation in all gene copies, dihydrolflate is inhibitory, while NADPH is not. A mutant containing only the Q67H substitution in all four gene copies is severely inhibited by both NADPH and substrate
additional information
construction of mutants based on mutant M1A/M16N/M20L/M42Y/C85A/M92F/C152S, i.e. ANLYF. Linear ANLYF-Gn is ANLYF with the N- and C-terminal extensions of Ala-(Gly)n-3 and -(Gly)2-Cys-Ala residues, respectively, with n=6-8, and circular ANLYF-Gn is ANLYF with the C and N termini cross-linked by the linker of the -Gly-Gly-Ala-(Gly)n-3 sequence, n=6-8
additional information
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construction of mutants based on mutant M1A/M16N/M20L/M42Y/C85A/M92F/C152S, i.e. ANLYF. Linear ANLYF-Gn is ANLYF with the N- and C-terminal extensions of Ala-(Gly)n-3 and -(Gly)2-Cys-Ala residues, respectively, with n=6-8, and circular ANLYF-Gn is ANLYF with the C and N termini cross-linked by the linker of the -Gly-Gly-Ala-(Gly)n-3 sequence, n=6-8
additional information
competitive hydrogen to deuterium and hydrogen to tritium kinetic isotope effects (KIEs) on the second-order rate constant (kcat/Km) are measured for wild-type and mutant ecDHFR variants to assess the intrinsic KIE on the catalyzed hydride transfer
additional information
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competitive hydrogen to deuterium and hydrogen to tritium kinetic isotope effects (KIEs) on the second-order rate constant (kcat/Km) are measured for wild-type and mutant ecDHFR variants to assess the intrinsic KIE on the catalyzed hydride transfer
additional information
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preparation of a dimeric variant (Xet-3) of dihydrofolate reductase from Escherichia coli by introducing residues located at the Thermotoga maritima dihydrofolate reductase (TmDHFR) dimer interface increases the melting temperature to about 60°C, approximately 9°C higher than that measured for the Escherichia coli enzyme. The steady-state and pre-steady-state rate constants measured for Xet-3 were similar to those of dimeric TmDHFR but significantly lower than those of the parent dihydrofolate reductase from Escherichia coli
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replacement of an integral alpha-helical segment of Escherichia coli DHFR with segments from a number of different organisms (Listeria grayi, Haemophilus influenzae, Streptococcus dysgalactiae, Saccharomyces cerevisiae, Plasmodium falciparum, Homo sapiens). The parameters most sensitive to helical replacement are KM and Ki. The rank orders of KM, Ki, change of Tm, and protein stability against unfolding to the molten-globule state are significantly correlated with helical content
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replacement of an integral alpha-helical segment of Escherichia coli DHFR with segments from a number of different organisms (Listeria grayi, Haemophilus influenzae, Streptococcus dysgalactiae, Saccharomyces cerevisiae, Plasmodium falciparum, Homo sapiens). The parameters most sensitive to helical replacement are KM and Ki. The rank orders of KM, Ki, change of Tm, and protein stability against unfolding to the molten-globule state are significantly correlated with helical content
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Halalkalibacterium halodurans
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alkaliphilic properties of enzyme are diminished upon addition of a N-terminal His-tag
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Halalkalibacterium halodurans
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a C-terminal His-tag does not affect the catalytic properties of the enzyme, but results in loss of the thermal stability and of the alkali-stable phenotype
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Halalkalibacterium halodurans C-125
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alkaliphilic properties of enzyme are diminished upon addition of a N-terminal His-tag
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Halalkalibacterium halodurans C-125
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a C-terminal His-tag does not affect the catalytic properties of the enzyme, but results in loss of the thermal stability and of the alkali-stable phenotype
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construction of hdrAgene, hdrB gene and double deletion mutants
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19-base pair deletion polymorphism is not associated with overall breast cancer risk
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analysis of intronic 19-bp deletion polymorphism and two polymorphisms within the 3' untranslated region as candidates for risk of neural tube defect. The 829C>T polymorphism is not found to be variable within the Irish population. The 19-bp intron deletion and the 721A>T polymorphisms are found to be in linkage disequilibrium. The 19-bp intron deletion allele shows a significant protective effect in mothers of neural tube defect cases when present in one or two copies
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characterization of ten methotrexate-resistant mutants with different amino acid combinations at residues F31, F34, and Q35. Higher resistance roughly correlates with a greater number of mutations, and an inverse correlation is observed between resistance and catalytic efficiency. Methotrexate-resistant dihydrofolate reductase can protect eucaryotic cells from methotrexate toxicity after transfection into dihydrofolate reductase knock-out cells
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characterization of ten methotrexate-resistant mutants with different amino acid combinations at residues F31, F34, and Q35. Higher resistance roughly correlates with a greater number of mutations, and an inverse correlation is observed between resistance and catalytic efficiency. Methotrexate-resistant dihydrofolate reductase can protect eucaryotic cells from methotrexate toxicity after transfection into dihydrofolate reductase knock-out cells
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streamlined, 2-tier selection and screening protocol to rapidly identify enzyme variants by saturation mutagenesis of active-site residues 7, 15, 24, 70, and 115, each known or suspected to affect ligand binding, The five libraries are selected for conservation of native-like activity and for resistance against methotrexate
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study on the association of polymorphisms in dihydrofolate reductase gene and treatment response in children with acute lymphoblastic leukemia. Lower event-free survival is associated with homozygosity for the allele A-317 and C-1610, and with the haplotype *1, and mRNA analysis shows higher dihydrofolate reductase levels for haplotype *1 carriers
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C-terminal-truncated mutant with lower activity
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knockout mutant dhfr-ts-
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selection of cell line with altered enzyme due to decreased methotrexate-sensitivity
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transformation of murine dihydrofolate reductase cDNA by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR, which differs from the murine sequence by 19 amino acids. Expression of the bovine dihydrofolate reductase cDNA in bacterial cells produces a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein
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incorporation of an alkyne-bearing non-natural amino acid, p-ethynylphenylalanine, into murine dihydrofolate reductase (mDHFR) at Val43, in high cell density cultivation of Escherichia coli, and performance of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation with fluorescent azide dyes to evaluate enzyme compatibility with various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19fold based on the same amount of non-natural amino acid. Enzyme incubation under optimized reaction condition does not lead to any activity loss but allows a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer is efficiently conjugated to enzyme mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. Method evaluation, overview
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incorporation of an alkyne-bearing non-natural amino acid, p-ethynylphenylalanine, into murine dihydrofolate reductase (mDHFR) at Val43, in high cell density cultivation of Escherichia coli, and performance of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation with fluorescent azide dyes to evaluate enzyme compatibility with various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19fold based on the same amount of non-natural amino acid. Enzyme incubation under optimized reaction condition does not lead to any activity loss but allows a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer is efficiently conjugated to enzyme mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. Method evaluation, overview
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sequence analysis of enzyme isolated from strains in different regions of India. Prevalence of double enzyme mutants and some with triple mutants in isolates from mainland, presence of qudruple mutants in isolates from Car Nicobar Island. Association between the degree of malaria transmission and the number of enzyme mutations
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construction of 19 mutants of residue W48. All mutants show very low specific activites
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deletion mutant lacking residues 2-5 of N-terminal tail compared to an analogous deletion of residues 2-22 of the N-terminal tail and construction of a mutant in the crossover helix that interacts with the dihydrofolate reductase active site by substitution of its five residues with alanines to form the Ala-FACE helix mutant. Mutations to the linker region within the bifunctional thymidylate synthase-dihydrofolate reductase affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the dihydrofolate reductase single rate and the bifunctional thymidylate synthase-dihydrofolate reductase rate by a factor of 2. The 2-fold activation of the dihydrofolate reductase rate by thymidylate synthase ligands remains intact, although even the activated N-terminal mutant has just half the dihydrolfolate reductase activity of the wild-type enzyme. However, the reciprocal communication between thymidlyate synthase active site and dihydrolfolate reductase ligands is impaired in N-terminal mutants
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deletion mutant lacking residues 2-5 of N-terminal tail compared to an analogous deletion of residues 2-22 of the N-terminal tail and construction of a mutant in the crossover helix that interacts with the dihydrofolate reductase active site by substitution of its five residues with alanines to form the Ala-FACE helix mutant. Mutations to the linker region within the bifunctional thymidylate synthase-dihydrofolate reductase affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the dihydrofolate reductase single rate and the bifunctional thymidylate synthase-dihydrofolate reductase rate by a factor of 2. The 2-fold activation of the dihydrofolate reductase rate by thymidylate synthase ligands remains intact, although even the activated N-terminal mutant has just half the dihydrolfolate reductase activity of the wild-type enzyme. However, the reciprocal communication between thymidlyate synthase active site and dihydrolfolate reductase ligands is impaired in N-terminal mutants
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heteroduplex tracking assay to detect dihydrofolate redctase L164-mutations in variants representing 1% of the parasites in an individual host
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pyrimethamine-resistant quadruple mutant 51I/59R/108N/213A, kcat value 0.68 per s, Km value for dihydrofolate, 0.0102 mM, Km value for NADPH, 0.0309 mM
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study on Plasmodium falciparum isolates from 118 children in Cote d'Ivoire. 39.5% are highly resistant to pyrimethamine, with IC50 values above 2000 nM. 39% of the isolates have mutant dihydrofolate reductase and 94% mutant dihydropteroate synthetase genes, and mutant dihydrofolate reductase is associated with resistance to pyrimethamine in vivo and in vitro
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deletion mutant lacking residues 2-5 of N-terminal tail compared to an analogous deletion of residues 2-22 of the N-terminal tail and construction of a mutant in the crossover helix that interacts with the dihydrofolate reductase active site by substitution of its five residues with alanines to form the Ala-FACE helix mutant. Mutations to the linker region within the bifunctional thymidylate synthase-dihydrofolate reductase affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the dihydrofolate reductase single rate and the bifunctional thymidylate synthase-dihydrofolate reductase rate by a factor of 2. The 2-fold activation of the dihydrofolate reductase rate by thymidylate synthase ligands remains intact, although even the activated N-terminal mutant has just half the dihydrolfolate reductase activity of the wild-type enzyme. However, the reciprocal communication between thymidlyate synthase active site and dihydrolfolate reductase ligands is impaired in N-terminal mutants
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survey of polymorphisms in 35 isolates of Plasmodium malariae collected from five countries in Asia and Africa reveals a low copy number of nonsynonymous mutations in five codons
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analysis of polymorphisms among 129 isolates from different geographical areas in India. A gradual increase in the frequency of mutant genotypes is observed from north to south, while isolates from the Car-Nicobar islands show only mutant genotypes
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expression of mutant AMRU1, i.e. 57L/58R/61M/1117T, in Plasmodium falciparum provides significant protection against pyrimethamine, cycloguanil, and clociguanil. It confers greater resistance to cycloguanil, clociguanil, and WR99210 than the homologous Plasmodium falciparum mutant
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expression of mutant AMRU1, i.e. 57L/58R/61M/1117T, in Plasmodium falciparum provides significant protection against pyrimethamine, cycloguanil, and clociguanil. It confers greater resistance to cycloguanil, clociguanil, and WR99210 than the homologous Plasmodium falciparum mutant
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DHFRLS in wild black rat has an intact open reading frame due to a 2 bp deletion compared to the brown rat or the Brown Norway rat
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DHFRLS in wild black rat has an intact open reading frame due to a 2 bp deletion compared to the brown rat or the Brown Norway rat
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a helix deletion reduces the DHFR catalytic efficiency by 30fold
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a helix deletion reduces the DHFR catalytic efficiency by 30fold
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growth of a single knock-out line of bifunctional dihydrofolate reductase-thymidylate synthase is identical to wild-type cells. Double knock-out cells have an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. Double knock-out lines are unable to infect mice, whereas the virulence of single knock-out lines is similar to wild-type. Double knock-out trypanosomes show reduced sensitivity to trimetrexate or raltitrexed. Pteridine reductase is not able to compensate for loss of dihydrofolate activity
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DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview
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DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview
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DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview
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