Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids. While the functional dihydrofolate reductase gene has been mapped to chromosome 5, multiple intronless processed pseudogenes or dihydrofolate reductase-like genes have been identified on separate chromosomes.
DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate. In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to NADP+. The high flexibility of Met20 and other loops near the active site play a role in promoting the release of the product, tetrahydrofolate. In particular the Met20 loop helps stabilize the nicotinamide ring of the NADPH to promote the transfer of the hydride from NADPH to dihydrofolate.
The reduction of dihydrofolate to tetrahydrofolate.
Found in all organisms, DHFR has a critical role in regulating the amount of tetrahydrofolate in the cell. Tetrahydrofolate and its derivatives are essential for purine and thymidylate synthesis, which are important for cell proliferation and cell growth. DHFR plays a central role in the synthesis of nucleic acid precursors, and it has been shown that mutant cells that completely lack DHFR require glycine, an amino acid, and thymidine to grow. DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin 
Dihydrofolate reductase deficiency has been linked to megaloblastic anemia. Treatment is with reduced forms of folic acid. Because tetrahydrofolate, the product of this reaction, is the active form of folate in humans, inhibition of DHFR can cause functional folate deficiency. Its central role in DNA precursor synthesis, coupled with its inhibition by antagonists such as trimethoprim and methotrexate, which are used as anti-bacterial or anti-cancer agents, has made DHFR a target of anticancer chemotherapy. However, resistance has developed against some drugs, as a result of changes in DHFR itself.
Therapeutic application and disease relevance
Since folate is needed by rapidly dividing cells to make thymine, this effect may be used to therapeutic advantage.
DHFR can be targeted in the treatment of cancer. DHFR is responsible for the levels of tetrahydrofolate in a cell, and the inhibition of DHFR can limit the growth and proliferation of cells that are characteristic of cancer. Methotrexate, a competitive inhibitor of DHFR, is one such anticancer drug that inhibits DHFR. Other drugs include trimethoprim and pyrimethamine. These three are widely used as antitumor and antimicrobial agents. Whether or not these are potent anticancer agents is unclear.
Trimethoprim has shown to have activity against a variety of Gram-positive bacterial pathogens. However, resistance to trimethoprim and other drugs aimed at DHFR can arise due to a variety of mechanisms, limiting the success of their therapeutical uses. Resistance can arise from DHFR gene amplification, mutations in DHFR, decrease in the uptake of the drugs, among others. Regardless, trimethoprim and sulfamethoxazole in combination has been used as an antibacterial agent for decades.
Folic acid is necessary for growth, and the pathway of the metabolism of folic acid is a target in developing treatments for cancer. DHFR is one such target. A regimen of fluorouracil, doxorubicin, and methotrexate was shown to prolong survival in patients with advanced gastric cancer. Further studies into inhibitors of DHFR can lead to more ways to treat cancer.
Dihydrofolate reductase as a target in the treatment of anthrax
Structural alignment of dihydrofolate reductase from Bacillus anthracis (BaDHFR), Staphylococcus aureus (SaDHFR), Escherichia coli (EcDHFR), and Streptococcus pneumoniae (SpDHFR).
BaDHFR's resistance to trimethoprim analogs is due to these two residues (F96 and Y102), which also confer improved kinetics and catalytic efficiency. Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.
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