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Dihydrofolate reductase, or DHFR, is an
enzyme that reduces
dihydrofolic acid to
tetrahydrofolic acid, using
NADPH as
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 DHFRgene.[1][2]
It is found in the q11→q22 region of chromosome 5.[3] Bacterial
species possess distinct DHFR
enzymes (based on their pattern of binding diaminoheterocyclic molecules), but
mammalian DHFRs are highly similar.[4]
Structure
A central eight-stranded
beta-pleated sheet makes up the main feature of the
polypeptide backbone folding of DHFR.[5] Seven of these strands are parallel and the eighth runs antiparallel. Four
alpha helices connect successive beta strands.[6] Residues 9 – 24 are termed "Met20" or "loop 1" and, along with other loops, are part of the major subdomain that surround the
active site.[7] The
active site is situated in the
N-terminal half of the sequence, which includes a
conservedPro-
Trp dipeptide; the
tryptophan has been shown to be involved in the binding of
substrate by the enzyme.[8]
Human DHFR with bound dihydrofolate and NADPH
Function
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.[9]
Reaction catalyzed by DHFR.
Tetrahydrofolate synthesis pathway.
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.[10] 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.[11] DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin[12]
Mechanism
The reduction of dihydrofolate to tetrahydrofolate.
Since DHFR serves as an important model for mechanistic studies in enzymology, its catalytic mechanisms with different substrates have been investigated for a while using a wide range of methods including X-ray, NMR structures, molecular dynamics simulation, enzyme kinetic measurements, Raman spectroscopy analysis, and ensemble and single-molecule kinetics.[13][14][15][16][17][18]
DHFR catalyzes the transfer of a hydride from
NADPH to
dihydrofolate with an accompanying protonation to produce
tetrahydrofolate.[10] 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.[7]
The catalytic cycle of the reaction catalyzed by DHFR incorporates five important intermediate: holoenzyme (E:NADPH), Michaelis complex (E:NADPH:DHF), ternary product complex (E:NADP+:THF), tetrahydrofolate binary complex (E:THF), and THF‚NADPH complex (E:NADPH:THF). The kinetic studies showed that the product (THF) dissociation step from E:NADPH:THF to E:NADPH is the rate determining step during steady-state turnover.[19]
Conformational changes are critical in DHFR's catalytic mechanism.[13] The Met20 loop of DHFR is able to open, close or occlude the active site.[20][21] Correspondingly, three different conformations classified as the opened, closed and occluded states are assigned to Met20. In addition, an extra distorted conformation of Met20 was defined due to its indistinct characterization results.[21] The Met20 loop is observed in its occluded conformation in the three product ligating intermediates, where the nicotinamide ring is occluded from the active site. This conformational feature accounts for the fact that the substitution of NADP+ by NADPH is prior to product dissociation. Thus, the next round of reaction can occur upon the binding of substrate.[19]
EcDHFR in ternary complex with NADPH and the substrate
Studies on the kinetic mechanism of dehydrofolate reductase from Mycobacterium tuberculosis and Escherichia coli indicate that the mechanism of this enzyme is stepwise and steady-state random. Specifically, the catalytic reaction begins with the NADPH and the substrate attaching to the binding site of the enzyme, followed by the protonation and the hydride transfer from the cofactor NADPH to the substrate. However, two latter steps do not take place simultaneously in a same transition state.[20][22] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.[23]
DHFR's enzymatic mechanism is shown to be pH dependent, particularly the hydride transfer step, since pH changes are shown to have remarkable influence on the electrostatics of the active site and the ionization state of its residues.[23] The acidity of the targeted nitrogen on the substrate is important in the binding of the substrate to the enzyme's binding site which is proved to be hydrophobic even though it has direct contact to water.[20][24] Asp27 is the only charged hydrophilic residue in the binding site, and neutralization of the charge on Asp27 may alter the pKa of the enzyme. Mutagenesis studies on important side chains show that Asp27 may play a critical role in the catalytic mechanism by helping with protonation of the substrate and restraining the substrate in the conformation favorable for the hydride transfer.[19][24][20] The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.[22] In some other studies, a water molecule is proved to be involved in the protonation step[21][17][25]. Analysis of DHFR crystal structures as well as simulation studies have proved that the entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.[15]
Clinical significance
Dihydrofolate reductase deficiency has been linked to
megaloblastic anemia.[9] 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. DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor synthesis.
Trimethoprim, an
antibiotic, inhibits bacterial DHFR while
methotrexate, a
chemotherapy agent, inhibits mammalian DHFR. However,
resistance has developed against some drugs, as a result of mutational changes in DHFR itself.[26]
DHFR mutations cause a rare autosomal recessive inborn error of folate metabolism that results in
megaloblastic anemia,
pancytopenia and severe cerebral folate deficiency which can be corrected by
folinic acid supplementation .[27]
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.[28] Other drugs include
trimethoprim and
pyrimethamine. These three are widely used as antitumor and antimicrobial agents.[29]
Trimethoprim has shown to have activity against a variety of
Gram-positive bacterial pathogens.[30] 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.[31][32][33] 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.[30]
Folic acid is necessary for growth,[34] 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.[35] Further studies into inhibitors of DHFR can lead to more ways to treat cancer.
Bacteria also need DHFR to grow and multiply and hence inhibitors selective for bacterial DHFR have found application as antibacterial agents.[30]
Classes of small-molecules employed as inhibitors of dihydrofolate reductase include diaminoquinazoline & diaminopyrroloquinazoline,[36] diaminopyrimidine,
diaminopteridine and diaminotriazines.[37]
Potential anthrax treatment
Structural alignment of dihydrofolate reductase from Bacillus anthracis (BaDHFR), Staphylococcus aureus (SaDHFR), Escherichia coli (EcDHFR), and Streptococcus pneumoniae (SpDHFR).
Dihydrofolate reductase from
Bacillus anthracis (BaDHFR) 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.[38] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.[38]
DHFR lacking
CHO cells are the most commonly used
cell line for the production of recombinant proteins. These cells are
transfected with a
plasmid carrying the dhfr gene and the gene for the recombinant protein in a single
expression system, and then subjected to
selective conditions in thymidine-lacking
medium. Only the cells with the exogenous DHFR gene along with the gene of interest survive.
Interactions
Dihydrofolate reductase has been shown to interact with
GroEL[39] and
Mdm2.[40]
Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles.[§ 1]
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^
abOsborne MJ, Schnell J, Benkovic SJ, Dyson HJ, Wright PE (August 2001). "Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism". Biochemistry. 40 (33): 9846–59.
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abSchnell JR, Dyson HJ, Wright PE (2004). "Structure, dynamics, and catalytic function of dihydrofolate reductase". Annual Review of Biophysics and Biomolecular Structure. 33 (1): 119–40.
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^Li R, Sirawaraporn R, Chitnumsub P, Sirawaraporn W, Wooden J, Athappilly F, Turley S, Hol WG (January 2000). "Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs". Journal of Molecular Biology. 295 (2): 307–23.
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^Benkovic SJ, Fierke CA, Naylor AM (March 1988). "Insights into enzyme function from studies on mutants of dihydrofolate reductase". Science. 239 (4844): 1105–10.
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abcHawser S, Lociuro S, Islam K (March 2006). "Dihydrofolate reductase inhibitors as antibacterial agents". Biochemical Pharmacology. 71 (7): 941–8.
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^Narayana N, Matthews DA, Howell EE, Nguyen-huu X (November 1995). "A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site". Nature Structural Biology. 2 (11): 1018–25.
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