A central eight-stranded beta-pleated sheet makes up the main feature of the polypeptide backbone folding of DHFR.

[16] The active site is situated in the N-terminal half of the sequence, which includes a conserved Pro-Trp dipeptide; the tryptophan has been shown to be involved in the binding of substrate by the enzyme.

[15] DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate.

The high flexibility of Met20 and other loops near the active site play a role in promoting the release of the product, tetrahydrofolate.

[17][18] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.

[28] Compare with E. coli chromosomal DHFR, it has higher Km in binding dihydrofolate (DHF) and NADPH.

The much lower catalytical kinetics show that hydride transfer is the rate determine step rather than product (THF) release.

[10][31][32] DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor (thymine) synthesis.

A regimen of fluorouracil, doxorubicin, and methotrexate was shown to prolong survival in patients with advanced gastric cancer.

[37] 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.

Dihydrofolate reductase from Bacillus anthracis (BaDHFR) is a validated drug target in the treatment of the infectious disease, anthrax.

BaDHFR is less sensitive to trimethoprim analogs than is dihydrofolate reductase from other species such as Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae.

A structural alignment of dihydrofolate reductase from all four species shows that only BaDHFR has the combination phenylalanine and tyrosine in positions 96 and 102, respectively.

BaDHFR's resistance to trimethoprim analogs is due to these two residues (F96 and Y102), which also confer improved kinetics and catalytic efficiency.

[44] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.

[44] DHFR has been used as a tool to detect protein–protein interactions in a protein-fragment complementation assay (PCA), using a split-protein approach.