Dihydrofolate reductase, or DHFR, is an
enzyme that reduces
dihydrofolic acid to
tetrahydrofolic acid, using
NADPH as an
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.[5][6] It is found in the q14.1 region of chromosome 5.[7]
There are two structural classes of DHFR, evolutionarily unrelated to each other. The former is usually just called DHFR and is found in bacterial chromosomes and animals. In bacteria, however, antibiotic pressure has caused this class to evolve different patterns of binding diaminoheterocyclic molecules, leading to many "types" named under this class, while
mammalian ones remain highly similar.[8] The latter (type II), represented by the plastid-encoded R67, is a tiny enzyme that works by forming a homotetramer.[9]
Dihydrofolate reductase converts
dihydrofolate into
tetrahydrofolate, a proton 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.[10]
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.[11] 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, a purine, and thymidine to grow.[12] DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin[13]
Structure
Dihydrofolate reductase
Crystal structure of chicken liver dihydrofolate reductase. PDB entry 8dfr
A central eight-stranded
beta-pleated sheet makes up the main feature of the
polypeptide backbone folding of DHFR.[14] Seven of these strands are parallel and the eighth runs antiparallel. Four
alpha helices connect successive beta strands.[15] 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.[16] 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.[15]
Mechanism
General mechanism
DHFR catalyzes the transfer of a hydride from
NADPH to
dihydrofolate with an accompanying protonation to produce
tetrahydrofolate.[11] 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.[16]
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.[17][18] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.[19]
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.[19] 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.[17][20] 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. Asp27 plays 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.[21][17][20] The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.[18] A water molecule is proved to be involved in the protonation step.[22][23][24] Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.[25]
Conformational changes of DHFR
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 product (THF) dissociation step from E:NADPH:THF to E:NADPH is the rate determining step during steady-state turnover.[21]
Conformational changes are critical in DHFR's catalytic mechanism.[26] The Met20 loop of DHFR is able to open, close or occlude the active site.[23][17] 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.[23] 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.[21]
R67 DHFR
R67 dihydrofolate reductase
R67 in complex with DHF and NADP+, monomer. PDB entry 2rk1.
Due to its unique structure and catalytic features, R67 DHFR is widely studied. R67 DHFR is a type II R-plasmid-encoded DHFR without geneticay or structural relation to the E. coli chromosomal DHFR. It is a homotetramer that possesses the 222 symmetry with a single active site pore that is exposed to solvent.[27] This symmetry of active site results in the different binding mode of the enzyme: It can bind with two dihydrofolate (DHF) molecules with positive cooperativity or two NADPH molecules with negative cooperativity, or one substrate plus one, but only the latter one has the catalytical activity.[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.[29]
In the R67 DHFR structure, the homotetramer forms an active site pore. In the catalytical process, DHF and NADPH enters into the pore from opposite position. The π-π stacking interaction between NADPH's nicotinamide ring and DHF's pteridine ring tightly connect two reactants in the active site. However, the flexibility of p-aminobenzoylglutamate tail of DHF was observed upon binding which can promote the formation of the transition state.[30]
Reaction Kinetics comparison between E. coli DHFR (EcDHFR) and R67 DHFR
Structure difference of substrate binding in EcDHFR and R67 DHFR
DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor (
thymine) 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.[33]
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 and bacterial infections.
Methotrexate, a
competitive inhibitor of DHFR, is one such anticancer drug that inhibits DHFR.[34]
Folate is necessary for growth,[35] and the pathway of the metabolism of folate 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.[36] Further studies into inhibitors of DHFR can lead to more ways to treat cancer.
Infection
Bacteria also need DHFR to grow and multiply and hence inhibitors selective for bacterial DHFR have found application as antibacterial agents.[37] Trimethoprim has shown to have activity against a variety of
Gram-positive bacterial pathogens.[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.[38][39][40] Resistance can arise from DHFR gene amplification,
mutations in DHFR,[41][42] decrease in the uptake of the drugs, among others. Regardless, trimethoprim and
sulfamethoxazole in combination has been used as an antibacterial agent for decades.[37]
Other classes of compounds that target DHFR in general, and bacterial DHFRs in particular, belong to the classes such as diaminopteridines, diaminotriazines, diaminopyrroloquinazolines, stilbenes, chalcones, deoxybenzoins, diaminoquinazolines, diaminopyrroloquinazolines, to name but a few.
Potential anthrax treatment
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-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. Supplementation of this medium with methotrexate, a competitive inhibitor of DHFR, can further select for those cells expressing the highest levels of DHFR, and thus, select for the top recombinant protein producers.[46]
Interactions
Dihydrofolate reductase has been shown to interact with
GroEL[47] and
Mdm2.[48]
Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles.[§ 1]
^Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K (July 1977). "Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate". Science. 197 (4302): 452–5.
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^Reyes VM, Sawaya MR, Brown KA, Kraut J (February 1995). "Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications". Biochemistry. 34 (8): 2710–23.
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abcSawaya MR, Kraut J (January 1997). "Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence". Biochemistry. 36 (3): 586–603.
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^Chen YQ, Kraut J, Blakley RL, Callender R (June 1994). "Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications". Biochemistry. 33 (23): 7021–6.
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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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^Bradrick TD, Beechem JM, Howell EE (September 1996). "Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein". Biochemistry. 35 (35): 11414–24.
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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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^Ng SK (2012). "Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker". Protein Expression in Mammalian Cells. Methods in Molecular Biology. Vol. 801. pp. 161–172.
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^Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P, Hartl FU (February 1996). "Protein folding in the central cavity of the GroEL-GroES chaperonin complex". Nature. 379 (6564): 420–6.
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Chan DC, Fu H, Forsch RA, Queener SF, Rosowsky A (June 2005). "Design, synthesis, and antifolate activity of new analogues of piritrexim and other diaminopyrimidine dihydrofolate reductase inhibitors with omega-carboxyalkoxy or omega-carboxy-1-alkynyl substitution in the side chain". Journal of Medicinal Chemistry. 48 (13): 4420–31.
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Bibcode:
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Cody V, Galitsky N, Luft JR, Pangborn W, Rosowsky A, Blakley RL (November 1997). "Comparison of two independent crystal structures of human dihydrofolate reductase ternary complexes reduced with nicotinamide adenine dinucleotide phosphate and the very tight-binding inhibitor PT523". Biochemistry. 36 (45): 13897–903.
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