Dihydrofolate reductase

Dihydrofolate reductase, or DHFR, is an is_associated_with::enzyme that reduces is_associated_with::dihydrofolic acid to is_associated_with::tetrahydrofolic acid, using is_associated_with::NADPH as is_associated_with::electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the DHFR is_associated_with::gene. It is found in the q11→q22 region of chromosome 5. is_associated_with::Bacterial is_associated_with::species possesses distinct DHFR is_associated_with::enzymes (based on their pattern of binding diaminoheterocyclic molecules), but is_associated_with::mammalian DHFRs are highly similar.

Structure
A central eight-stranded is_associated_with::beta-pleated sheet makes up the main feature of the is_associated_with::polypeptide backbone folding of DHFR. Seven of these strands are parallel and the eighth runs antiparallel. Four is_associated_with::alpha helices connect successive beta strands. Residues 9 – 24 are termed “Met20” or “loop 1” and, along with other loops, are part of the major subdomain that surround the is_associated_with::active site. The is_associated_with::active site is situated in the is_associated_with::N-terminal half of the sequence, which includes a conserved Pro-Trp dipeptide; the is_associated_with::tryptophan has been shown to be involved in the binding of substrate by the enzyme.

Function
Dihydrofolate reductase converts is_associated_with::dihydrofolate into is_associated_with::tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of is_associated_with::purines, thymidylic acid, and certain is_associated_with::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.

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 is_associated_with::purine and is_associated_with::thymidylate synthesis, which are important for cell proliferation and cell growth. DHFR plays a central role in the synthesis of is_associated_with::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

Mechanism
DHFR catalyzes the transfer of a hydride from is_associated_with::NADPH to is_associated_with::dihydrofolate with an accompanying protonation to produce is_associated_with::tetrahydrofolate. In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to is_associated_with::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.

Clinical significance
Dihydrofolate reductase deficiency has been linked to is_associated_with::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 is_associated_with::folate deficiency. DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor synthesis. is_associated_with::Trimethoprim, an is_associated_with::antibiotic, inhibits bacterial DHFR while is_associated_with::methotrexate, a is_associated_with::chemotherapy agent, inhibits mammalian DHFR. However, resistance has developed against some drugs, as a result of mutational changes in DHFR itself.

Therapeutic applications
Since folate is needed by rapidly dividing cells to make is_associated_with::thymine, this effect may be used to therapeutic advantage.

DHFR can be targeted in the treatment of is_associated_with::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. is_associated_with::Methotrexate, a is_associated_with::competitive inhibitor of DHFR, is one such anticancer drug that inhibits DHFR. Other drugs include is_associated_with::trimethoprim and is_associated_with::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 is_associated_with::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, is_associated_with::mutations in DHFR, decrease in the uptake of the drugs, among others. Regardless, trimethoprim and is_associated_with::sulfamethoxazole in combination has been used as an antibacterial agent for decades.

is_associated_with::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 is_associated_with::fluorouracil, is_associated_with::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.

Potential anthrax treatment
Dihydrofolate reductase from is_associated_with::Bacillus anthracis (BaDHFR) a validated drug target in the treatment of the infectious disease, anthrax. BaDHFR is less sensitive to is_associated_with::trimethoprim analogs than is dihydrofolate reductase from other species such as is_associated_with::Escherichia coli, is_associated_with::Staphylococcus aureus, and is_associated_with::Streptococcus pneumoniae. A structural alignment of dihydrofolate reductase from all four species shows that only BaDHFR has the combination is_associated_with::phenylalanine and is_associated_with::tyrosine in positions 96 and 102, respectively.

BaDHFR's resistance to is_associated_with::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.

As a research tool
DHFR has been used as a tool to detect is_associated_with::protein-protein interactions in a is_associated_with::protein-fragment complementation assay (PCA).

Interactions
Dihydrofolate reductase has been shown to interact with is_associated_with::GroEL and is_associated_with::Mdm2.