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1.5.1.3: dihydrofolate reductase

This is an abbreviated version!
For detailed information about dihydrofolate reductase, go to the full flat file.

Word Map on EC 1.5.1.3

Reaction

5,6,7,8-tetrahydrofolate
+
NADP+
=
7,8-dihydrofolate
 +
NADPH
 +
H+

Synonyms

5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, 7,8-dihydrofolate reductase, At2g16370, At4g34570, bifunctional dihydrofolate reductase-thymidylate synthase, bifunctional TS-DHFR, BmDHFR, dehydrogenase, tetrahydrofolate, DFR-TS, DFR1, DfrA, DfrB, DHFR, DHFR type IIIC, DHFR-TS, DHFR-TS1, DHFR-TS2, DHFR2, DHFRL1, DHFRLS, dihydrofolate reductase, dihydrofolate reductase-like, dihydrofolate reductase-thymidylate synthase, dihydrofolate reductase:thymidylate synthase, dihydrofolic acid reductase, dihydrofolic reductase, EC 1.5.1.4, ecDHFR, folA, folA3, folic acid reductase, folic reductase, FolM, hDHFR, hDHFR-1, hDHFR-2, HjDHFR, hvDHFR1, hvDHFR2, LAU_0427, LBRM_06_0830, mDHFR, mjDHFR, More, myDHFR, NADPH-dihydrofolate reductase, pcDHFR, PKNH_0509600, ppDHFR, pteridine reductase, pteridine reductase:dihydrofolate reductase, PTR2, R-plasmid-encoded dihydrofolate reductase, R67 DHFR, R67 dihydrofolate reductase, reductase, dihydrofolate, S3DHFR, Smdhfr, Smp 175230, spDHFR, svDHFR, tcptr1, tetrahydrofolate dehydrogenase, THY-1, THY-2, thymidylate synthase-dihydrofolate reductase, thymidylate synthetase-dihydrofolate reductase, Trimethoprim resistance protein, TS-DHFR, WUBG_00817

ECTree

     1 Oxidoreductases
         1.5 Acting on the CH-NH group of donors
             1.5.1 With NAD+ or NADP+ as acceptor
                1.5.1.3 dihydrofolate reductase

Engineering

Engineering on EC 1.5.1.3 - dihydrofolate reductase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
G121V
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/N64F
Q35K/N64S
site-directed mutagenesis
Q35S/N64F
site-directed mutagenesis
Q35S/N64S
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
C59R/S108N
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
N51I/C59R/S108N/I164L
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
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
S108T
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
S58R/S117N
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
additional information