D6566
Dihydrofolate Reductase human
≥80% (SDS-PAGE), recombinant, expressed in E. coli, ≥1 units/mg protein
Synonym(s):
DHFR, Tetrahydrofolate NADP+ oxidoreductase
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About This Item
MDL number:
UNSPSC Code:
12352204
NACRES:
NA.54
recombinant
expressed in E. coli
Quality Level
Assay
≥80% (SDS-PAGE)
form
solution
specific activity
≥1 units/mg protein
mol wt
25 kDa
concentration
0.02-0.06 mg/mL
UniProt accession no.
shipped in
wet ice
storage temp.
−20°C
Gene Information
human ... DHFR(1719)
General description
Human DHFR is an 186 amino acid protein with an apparent molecular weight of 25 kDa. It is 30% homologous to the E. coli protein and up to 70% homologous to vertebrate proteins.
Application
Dihydrofolate Reductase human has been used:
- to investigate the stable expression of green fluorescent protein and the targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis
- to study the structural analysis of human dihydrofolate reductase as a binary complex
- to study its in vitro kinetic assay for the enzyme inhibition study
Human dihydrofolate reductase has been used in a study to investigate the stable expression of green fluorescent protein and the targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis. Human dihydrofolate reductase has also been used in a study to investigate the structural analysis of human dihydrofolate reductase as a binary complex.
Biochem/physiol Actions
Dihydrofolate reductase (DHFR) is a key enzyme in thymidine synthesis. It catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). At a much lower rate, it catayzes the conversion of folate to THF. Since thymidine is a necessary substrate for DNA synthesis, DHFR is a target for anticancer drug development. Methotrexate is the prototype dihydrofolate reductase inhibitor. The enzyme from Sigma has been used in the inhibitory studies of Leishmaniasis donovani pteridine reductase 1 (PTR1). The enzyme has also been used as a positive control to measure the DHFR activity of a protein, MS0308, purified from Mycobacterium smegmatis.
Km5,6
NADPH 0.16 mM
7,8-dihydrofolate 0.03 mM
8-methylpterin 0.13 mM
Ki7
Folate 2.6x10-5 mM
Methotrexate 6.1-9x10-9
NADPH 0.16 mM
7,8-dihydrofolate 0.03 mM
8-methylpterin 0.13 mM
Ki7
Folate 2.6x10-5 mM
Methotrexate 6.1-9x10-9
The human DHFR gene, as well as DHFR genes in other mammalian species, overcome the inhibitory effects of methotrexate by a mechanism of gene amplification or by amino-acid mutagenesis. Dihydroflate reductase (DHFR) catalyzes the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and, at a much lower rate, the conversion of folate to THF. The reaction product, THF, is an essential cofactor in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. It is a key enzyme in thymidine synthesis. Therefore, DHFR is a critical enzyme in DNA synthesis and has become a target for drug development and cancer therapy. The variations between DHFR from different sources have enabled the development of species selective DHFR inhibitors, such as trimethoprim (antibacterial and antifungal), pyrimethamine (antiprotozoal), and methotrexate; MTX (antineoplastic, antipsoriatic, and anti-inflammatory).
Unit Definition
One unit will convert 1.0 μmole of dihydrofolic acid to tetrahydrofolic acid in 1 minute at pH 7.5 at 22 °C.
Physical form
Solution in 10 mM Tris pH 8, 1 mM EDTA, 0.5 mM DTT, 5 μM NADPH, protease inhibitors, and 50% glycerol.
substrate
WGK
WGK 3
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Personal Protective Equipment
dust mask type N95 (US), Eyeshields, Gloves
Regulatory Information
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Dihydrofolate reductase: binding of substrates and inhibitors and catalytic mechanism.
J E Gready
Advances in pharmacology and chemotherapy, 17, 37-102 (1980-01-01)
Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse
Xu F, et al.
Febs Letters, 588, 3924-3931 (2014)
Eukaryotic dihydrofolate reductase.
R L Blakley
Advances in enzymology and related areas of molecular biology, 70, 23-102 (1995-01-01)
R Bryn Fenwick et al.
Proceedings of the National Academy of Sciences of the United States of America, 111(4), E445-E454 (2014-01-30)
Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes
Dimitrios Evangelopoulos et al.
The FEBS journal, 278(24), 4824-4832 (2011-10-07)
Mycobacterium tuberculosis, the most successful bacterial pathogen, causes tuberculosis, a disease that still causes more than 2 million deaths per year. Arylamine N-acetyltransferase is an enzyme that is conserved in most Mycobacterium spp. The nat gene belongs to an operon that
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