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D6566

Sigma-Aldrich

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

Enzyme Commission number:
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
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.

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US), Eyeshields, Gloves

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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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)
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
Eukaryotic dihydrofolate reductase.
R L Blakley
Advances in enzymology and related areas of molecular biology, 70, 23-102 (1995-01-01)
Masahito Asada et al.
Molecular and biochemical parasitology, 181(2), 162-170 (2011-11-24)
We have achieved stable expression of green fluorescent protein (GFP) in Babesia bovis by using the WR99210/human dihydrofolate reductase (DHFR) gene selection system. A GFP-expression plasmid with a dhfr expression cassette (DHFR-gfp) was constructed and transfected into B. bovis by

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