Editing Dihydrofolate reductase

{{Short description|Mammalian protein found in humans}}
{{cs1 config|name-list-style=vanc|display-authors=6}}
{{Use dmy dates|date=April 2016}}
{{Infobox_gene}}
'''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 [[cofactor (biochemistry)|cofactor]]s used in one-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the ''DHFR'' [[gene]].<ref name="pmid6961421">{{cite journal | vauthors = Chen MJ, Shimada T, Moulton AD, Harrison M, Nienhuis AW | title = Intronless human dihydrofolate reductase genes are derived from processed RNA molecules | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 79 | issue = 23 | pages = 7435–9 | date = December 1982 | pmid = 6961421 | pmc = 347354 | doi = 10.1073/pnas.79.23.7435 | bibcode = 1982PNAS...79.7435C | doi-access = free }}</ref><ref name="pmid6323448">{{cite journal | vauthors = Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW | title = The functional human dihydrofolate reductase gene | journal = The Journal of Biological Chemistry | volume = 259 | issue = 6 | pages = 3933–43 | date = March 1984 | doi = 10.1016/S0021-9258(17)43186-3 | pmid = 6323448 | doi-access = free }}</ref> It is found in the q14.1 region of chromosome 5.<ref>{{cite web |title=DHFR dihydrofolate reductase [Homo sapiens (human)] |url=https://www.ncbi.nlm.nih.gov/gene/1719 |website=Gene - NCBI |access-date=21 February 2023}}</ref>

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 [[mammal]]ian ones remain highly similar.<ref name="pmid500653">{{cite journal | vauthors = Smith SL, Patrick P, Stone D, Phillips AW, Burchall JJ | title = Porcine liver dihydrofolate reductase. Purification, properties, and amino acid sequence | journal = The Journal of Biological Chemistry | volume = 254 | issue = 22 | pages = 11475–84 | date = November 1979 | doi = 10.1016/S0021-9258(19)86510-9 | pmid = 500653 | doi-access = free }}</ref> The latter (type II), represented by the plastid-encoded R67, is a tiny enzyme that works by forming a homotetramer.<ref>{{cite journal | vauthors = Krahn JM, Jackson MR, DeRose EF, Howell EE, London RE | title = Crystal structure of a type II dihydrofolate reductase catalytic ternary complex | journal = Biochemistry | volume = 46 | issue = 51 | pages = 14878–14888 | date = December 2007 | pmid = 18052202 | pmc = 3743094 | doi = 10.1021/bi701532r }}</ref>

== Function ==
{{Infobox enzyme
| Name       = Dihydrofolate reductase
| EC_number  = 1.5.1.3
| CAS_number = 9002-03-3
| GO_code    = 0004146
}}
Dihydrofolate reductase converts [[dihydrofolate]] into [[tetrahydrofolate]], a proton shuttle required for the de novo synthesis of [[purines]], [[thymidine monophosphate|thymidylic acid]], and certain [[amino acid]]s. 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.<ref name="entrez">{{cite web | title = Entrez Gene: DHFR dihydrofolate reductase| url = https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1719}}</ref>

<gallery class="skin-invert-image" mode=packed>
Image:DHFR rxn.svg|Reaction catalyzed by DHFR.
Image:THFsynthesispathway.png|Tetrahydrofolate synthesis pathway.
</gallery>

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.<ref name="pmid15139807">{{cite journal | vauthors = Schnell JR, Dyson HJ, Wright PE | s2cid = 28611812 | title = Structure, dynamics, and catalytic function of dihydrofolate reductase | journal = Annual Review of Biophysics and Biomolecular Structure | volume = 33 | issue = 1 | pages = 119–40 | year = 2004 | pmid = 15139807 | doi = 10.1146/annurev.biophys.33.110502.133613 }}</ref> 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.<ref name="pmid6933469">{{cite journal | vauthors = Urlaub G, Chasin LA | title = Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 77 | issue = 7 | pages = 4216–20 | date = July 1980 | pmid = 6933469 | pmc = 349802 | doi = 10.1073/pnas.77.7.4216 | bibcode = 1980PNAS...77.4216U | doi-access = free }}</ref> DHFR has also been demonstrated as an enzyme involved in the salvage of [[tetrahydrobiopterin]] from [[dihydrobiopterin]].<ref>{{cite journal | vauthors = Crabtree MJ, Tatham AL, Hale AB, Alp NJ, Channon KM | title = Critical role for tetrahydrobiopterin recycling by dihydrofolate reductase in regulation of endothelial nitric-oxide synthase coupling: relative importance of the de novo biopterin synthesis versus salvage pathways | journal = The Journal of Biological Chemistry | volume = 284 | issue = 41 | pages = 28128–36 | date = October 2009 | pmid = 19666465 | pmc = 2788863 | doi = 10.1074/jbc.M109.041483 | doi-access = free }}</ref><ref>{{cite book|year=1989|title=Folates and Cobalamins|chapter=Chapter 17: The Relationship Between Biopterin and Folate Metabolism|vauthors=Harpey JP|pages=215-218|veditors=Zittoun JA, Cooper BA|isbn=978-3-540-50653-9|doi=10.1007/978-3-642-74364-1_17|publisher=Springer-Verlag}}</ref>

== Structure ==
{{Infobox protein family
| Symbol = DHFR_1
| Name = Dihydrofolate reductase
| image = PDB 8dfr EBI.jpg
| width = 
| caption = Crystal structure of chicken liver dihydrofolate reductase. PDB entry {{PDBe|8dfr}}
| Pfam = PF00186
| Pfam_clan = CL0387 
| InterPro = IPR001796
| SMART = 
| PROSITE = PDOC00072
| MEROPS = 
| SCOP = 1dhi
| TCDB = 
| OPM family = 
| OPM protein = 
| CAZy = 
| CDD = 
}}
A central eight-stranded [[beta-pleated sheet]] makes up the main feature of the [[polypeptide]] backbone folding of DHFR.<ref name="pmid17920">{{cite journal | vauthors = Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K | title = Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate | journal = Science | volume = 197 | issue = 4302 | pages = 452–5 | date = July 1977 | pmid = 17920 | doi = 10.1126/science.17920 | bibcode = 1977Sci...197..452M }}</ref> Seven of these strands are parallel and the eighth runs antiparallel. Four [[alpha helices]] connect successive beta strands.<ref name="pmid6815179">{{cite journal | vauthors = Filman DJ, Bolin JT, Matthews DA, Kraut J | title = Crystal structures of ''Escherichia coli'' and ''Lactobacillus casei'' dihydrofolate reductase refined at 1.7 Å resolution. II. Environment of bound NADPH and implications for catalysis | journal = The Journal of Biological Chemistry | volume = 257 | issue = 22 | pages = 13663–72 | date = November 1982 | doi = 10.1016/S0021-9258(18)33498-7 | pmid = 6815179 | doi-access = free }}</ref> 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]].<ref name="pmid11502178">{{cite journal|author-link4=Jane Dyson | vauthors = Osborne MJ, Schnell J, Benkovic SJ, Dyson HJ, Wright PE | title = Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism | journal = Biochemistry | volume = 40 | issue = 33 | pages = 9846–59 | date = August 2001 | pmid = 11502178 | doi = 10.1021/bi010621k }}</ref> The [[active site]] is situated in the [[N-terminal]] half of the sequence, which includes a [[conserved sequence|conserved]] [[Proline|Pro]]-[[Tryptophan|Trp]] dipeptide; the [[tryptophan]] has been shown to be involved in the binding of [[Enzyme substrate|substrate]] by the enzyme.<ref name="pmid6815179" />

== Mechanism ==

=== General mechanism ===
[[File:DHFR Reaction Scheme.png|thumb|left|class=skin-invert-image|The reduction of dihydrofolate to tetrahydrofolate catalyzed by DHFR]]
DHFR catalyzes the transfer of a hydride from [[NADPH]] to [[dihydrofolate]] with an accompanying protonation to produce [[tetrahydrofolate]].<ref name="pmid15139807" /> 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.<ref name="pmid11502178" />

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.<ref name="Rod_2003" /><ref name="Wan_2014">{{cite journal | vauthors = Wan Q, Bennett BC, Wilson MA, Kovalevsky A, Langan P, Howell EE, Dealwis C | title = Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 51 | pages = 18225–30 | date = December 2014 | pmid = 25453083 | pmc = 4280638 | doi = 10.1073/pnas.1415856111 | bibcode = 2014PNAS..11118225W | doi-access = free }}</ref> In a study using computational and experimental approaches, Liu ''et al'' conclude that the protonation step precedes the hydride transfer.<ref name="Liu_2014">{{cite journal | vauthors = Liu CT, Francis K, Layfield JP, Huang X, Hammes-Schiffer S, Kohen A, Benkovic SJ | title = ''Escherichia coli'' dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 51 | pages = 18231–6 | date = December 2014 | pmid = 25453098 | pmc = 4280594 | doi = 10.1073/pnas.1415940111 | bibcode = 2014PNAS..11118231L | doi-access = free }}</ref>

[[File:DHFR + NADPH + folate (Met20 loop).png|thumb|DHFR (Met20 loop highlighted) + NADPH + folate]]
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.<ref name="Liu_2014" /> 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.<ref name="Rod_2003" /><ref name="Czekster_2011">{{cite journal | vauthors = Czekster CM, Vandemeulebroucke A, Blanchard JS | title = Kinetic and chemical mechanism of the dihydrofolate reductase from ''Mycobacterium tuberculosis'' | journal = Biochemistry | volume = 50 | issue = 3 | pages = 367–75 | date = January 2011 | pmid = 21138249 | pmc = 3074011 | doi = 10.1021/bi1016843 }}</ref> 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.<ref name="Fierke_1987" /><ref name="Rod_2003" /><ref name="Czekster_2011" /> The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.<ref name="Wan_2014" /> A water molecule is proved to be involved in the protonation step.<ref name="Reyes_1995">{{cite journal | vauthors = Reyes VM, Sawaya MR, Brown KA, Kraut J | title = Isomorphous crystal structures of ''Escherichia coli'' dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications | journal = Biochemistry | volume = 34 | issue = 8 | pages = 2710–23 | date = February 1995 | pmid = 7873554 | doi = 10.1021/bi00008a039 }}</ref><ref name="Sawaya_1997" /><ref>{{cite journal | vauthors = Chen YQ, Kraut J, Blakley RL, Callender R | title = Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications | journal = Biochemistry | volume = 33 | issue = 23 | pages = 7021–6 | date = June 1994 | pmid = 8003467 | doi = 10.1021/bi00189a001 }}</ref> Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.<ref name="Shrimpton_2002">{{cite journal | vauthors = Shrimpton P, Allemann RK | title = Role of water in the catalytic cycle of E. coli dihydrofolate reductase | journal = Protein Science | volume = 11 | issue = 6 | pages = 1442–51 | date = June 2002 | pmid = 12021443 | pmc = 2373639 | doi = 10.1110/ps.5060102 }}</ref>

=== Conformational changes of DHFR ===
[[File:Conformational changes during the DHFR catalytic cycle.png|thumb|The closed structure is depicted in red and the occluded structure is depicted in green in the catalytic scheme. In the structure, DHF and THF are colored red, NADPH is colored yellow, and Met20 residue is colored blue.]]
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<sup href="NADP+">+</sup>: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.<ref name="Fierke_1987">{{cite journal | vauthors = Fierke CA, Johnson KA, Benkovic SJ | title = Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from ''Escherichia coli'' | journal = Biochemistry | volume = 26 | issue = 13 | pages = 4085–92 | date = June 1987 | pmid = 3307916 | doi = 10.1021/bi00387a052 }}</ref>

Conformational changes are critical in DHFR's catalytic mechanism.<ref>{{cite journal | vauthors = Antikainen NM, Smiley RD, Benkovic SJ, Hammes GG | title = Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase | journal = Biochemistry | volume = 44 | issue = 51 | pages = 16835–43 | date = December 2005 | pmid = 16363797 | doi = 10.1021/bi051378i }}</ref> The Met20 loop of DHFR is able to open, close or occlude the active site.<ref name="Sawaya_1997">{{cite journal | vauthors = Sawaya MR, Kraut J | title = Loop and subdomain movements in the mechanism of ''Escherichia coli'' dihydrofolate reductase: crystallographic evidence | journal = Biochemistry | volume = 36 | issue = 3 | pages = 586–603 | date = January 1997 | pmid = 9012674 | doi = 10.1021/bi962337c }}</ref><ref name="Rod_2003">{{cite journal | vauthors = Rod TH, Brooks CL | title = How dihydrofolate reductase facilitates protonation of dihydrofolate | journal = Journal of the American Chemical Society | volume = 125 | issue = 29 | pages = 8718–9 | date = July 2003 | pmid = 12862454 | doi = 10.1021/ja035272r }}</ref> 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.<ref name="Sawaya_1997" /> 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<sup>+</sup> by NADPH is prior to product dissociation. Thus, the next round of reaction can occur upon the binding of substrate.<ref name="Fierke_1987" />

=== R67 DHFR ===
{{Infobox protein family
| Symbol = DHFR_2
| Name = R67 dihydrofolate reductase
| image = PDB 2rk1 EBI.png
| width = 
| caption = R67 in complex with DHF and NADP+, monomer. PDB entry {{PDBe|2rk1}}.
| Pfam = PF06442
| Pfam_clan =  
| InterPro = IPR009159
| SMART = 
| PROSITE = 
| MEROPS = 
| SCOP = 1vif
| TCDB = 
| OPM family = 
| OPM protein = 
| CAZy = 
| CDD = 
}}
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.<ref>{{cite journal | vauthors = Narayana N, Matthews DA, Howell EE, Nguyen-huu X | title = A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site | journal = Nature Structural Biology | volume = 2 | issue = 11 | pages = 1018–25 | date = November 1995 | pmid = 7583655 | doi=10.1038/nsb1195-1018| s2cid = 11914241 }}</ref> 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.<ref>{{cite journal | vauthors = Bradrick TD, Beechem JM, Howell EE | title = 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 | journal = Biochemistry | volume = 35 | issue = 35 | pages = 11414–24 | date = September 1996 | pmid = 8784197 | doi = 10.1021/bi960205d }}</ref> Compare with E. coli chromosomal DHFR, it has higher K<sub>m</sub> 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.<ref>{{cite journal | vauthors = Park H, Zhuang P, Nichols R, Howell EE | title = Mechanistic studies of R67 dihydrofolate reductase. Effects of pH and an H62C mutation | journal = The Journal of Biological Chemistry | volume = 272 | issue = 4 | pages = 2252–8 | date = January 1997 | pmid = 8999931 | doi = 10.1074/jbc.272.4.2252 | doi-access = free }}</ref>

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]].<ref>{{cite journal | vauthors = Kamath G, Howell EE, Agarwal PK | title = The tail wagging the dog: insights into catalysis in R67 dihydrofolate reductase | journal = Biochemistry | volume = 49 | issue = 42 | pages = 9078–88 | date = October 2010 | pmid = 20795731 | doi = 10.1021/bi1007222 }}</ref>

<gallery class="skin-invert-image" mode=packed>
File:Reaction Kinetics comparison between EcDHFR and R67 DHFR.png|Reaction Kinetics comparison between ''E. coli'' DHFR (EcDHFR) and R67 DHFR
File:Structure difference of substrate binding in E. coli and R67 DHFR.png|Structure difference of substrate binding in EcDHFR and R67 DHFR
</gallery>

== Clinical significance ==
<!-- Human genetic issues only here. If you are targeting the enzyme, use the next section -->
DHFR mutations cause [[dihydrofolate reductase deficiency]], a rare autosomal recessive inborn error of folate metabolism that results in [[megaloblastic anemia]], [[pancytopenia]] and severe [[cerebral folate deficiency]]. These issues can be overcome by supplementation with a reduced form of folate, usually [[folinic acid]].<ref name="entrez"/><ref>{{cite journal | vauthors = Banka S, Blom HJ, Walter J, Aziz M, Urquhart J, Clouthier CM, Rice GI, de Brouwer AP, Hilton E, Vassallo G, Will A, Smith DE, Smulders YM, Wevers RA, Steinfeld R, Heales S, Crow YJ, Pelletier JN, Jones S, Newman WG | title = Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency | journal = American Journal of Human Genetics | volume = 88 | issue = 2 | pages = 216–25 | date = February 2011 | pmid = 21310276 | pmc = 3035707 | doi = 10.1016/j.ajhg.2011.01.004 }}</ref><ref name="NyhanHoffmann2011">{{cite book | vauthors = Nyhan WL, Hoffmann GF, Barshop BA |title=Atlas of Inherited Metabolic Diseases 3E|url=https://books.google.com/books?id=vCvSBQAAQBAJ&pg=PA141|date=30 December 2011|publisher=CRC Press|isbn=978-1-4441-4948-7|pages=141–}}</ref>

== Therapeutic applications ==
{{main|Dihydrofolate reductase inhibitor}}
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, [[Disease resistance|resistance]] has developed against some drugs, as a result of mutational changes in DHFR itself.<ref name="pmid2601715">{{cite journal | vauthors = Cowman AF, Lew AM | title = Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi | journal = Molecular and Cellular Biology | volume = 9 | issue = 11 | pages = 5182–8 | date = November 1989 | pmid = 2601715 | pmc = 363670 | doi = 10.1128/mcb.9.11.5182 }}</ref>

=== 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.<ref name="pmid10623528">{{cite journal | vauthors = Li R, Sirawaraporn R, Chitnumsub P, Sirawaraporn W, Wooden J, Athappilly F, Turley S, Hol WG | s2cid = 24527344 | title = Three-dimensional structure of ''M. tuberculosis'' dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs | journal = Journal of Molecular Biology | volume = 295 | issue = 2 | pages = 307–23 | date = January 2000 | pmid = 10623528 | doi = 10.1006/jmbi.1999.3328 }}</ref>

[[Folate]] is necessary for growth,<ref name="pmid19706381">{{cite journal | vauthors = Bailey SW, Ayling JE | title = The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 36 | pages = 15424–9 | date = September 2009 | pmid = 19706381 | pmc = 2730961 | doi = 10.1073/pnas.0902072106 | doi-access = free }}</ref> 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.<ref name="pmid8508427">{{cite journal | vauthors = Murad AM, Santiago FF, Petroianu A, Rocha PR, Rodrigues MA, Rausch M | title = Modified therapy with 5-fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer | journal = Cancer | volume = 72 | issue = 1 | pages = 37–41 | date = July 1993 | pmid = 8508427 | doi = 10.1002/1097-0142(19930701)72:1<37::AID-CNCR2820720109>3.0.CO;2-P | doi-access = free }}</ref> 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.<ref name="pmid16359642"/> Trimethoprim has shown to have activity against a variety of [[Gram-positive]] bacterial pathogens.<ref name="pmid16359642">{{cite journal | vauthors = Hawser S, Lociuro S, Islam K | title = Dihydrofolate reductase inhibitors as antibacterial agents | journal = Biochemical Pharmacology | volume = 71 | issue = 7 | pages = 941–8 | date = March 2006 | pmid = 16359642 | doi = 10.1016/j.bcp.2005.10.052 }}</ref> 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.<ref name="pmid7583655">{{cite journal | vauthors = Narayana N, Matthews DA, Howell EE, Nguyen-huu X | title = A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site | journal = Nature Structural Biology | volume = 2 | issue = 11 | pages = 1018–25 | date = November 1995 | pmid = 7583655 | doi = 10.1038/nsb1195-1018 | s2cid = 11914241 }}</ref><ref name="pmid8762155">{{cite journal | vauthors = Huennekens FM | title = In search of dihydrofolate reductase | journal = Protein Science | volume = 5 | issue = 6 | pages = 1201–8 | date = June 1996 | pmid = 8762155 | pmc = 2143423 | doi = 10.1002/pro.5560050626 }}</ref><ref name="pmid12084458">{{cite journal | vauthors = Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick R, Bertino JR | title = Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase | journal = Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease | volume = 1587 | issue = 2–3 | pages = 164–73 | date = July 2002 | pmid = 12084458 | doi = 10.1016/S0925-4439(02)00079-0 | doi-access = free }}</ref> Resistance can arise from DHFR gene amplification, [[mutations]] in DHFR,<ref>{{cite journal | vauthors = Toprak E, Veres A, Michel JB, Chait R, Hartl DL, Kishony R | title = Evolutionary paths to antibiotic resistance under dynamically sustained drug selection | journal = Nature Genetics | volume = 44 | issue = 1 | pages = 101–5 | date = December 2011 | pmid = 22179135 | pmc = 3534735 | doi = 10.1038/ng.1034 }}</ref><ref>{{cite journal | vauthors = Rodrigues JV, Bershtein S, Li A, Lozovsky ER, Hartl DL, Shakhnovich EI | title = Biophysical principles predict fitness landscapes of drug resistance | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 11 | pages = E1470-8 | date = March 2016 | pmid = 26929328 | pmc = 4801265 | doi = 10.1073/pnas.1601441113 | bibcode = 2016PNAS..113E1470R | doi-access = free }}</ref> decrease in the uptake of the drugs, among others. Regardless, trimethoprim and [[sulfamethoxazole]] in combination has been used as an antibacterial agent for decades.<ref name="pmid16359642"/>

[[Pyrimethamine]] is a widely used [[antiprotozoal]] agent.<ref name="pmid3125607">{{cite journal | vauthors = Benkovic SJ, Fierke CA, Naylor AM | title = Insights into enzyme function from studies on mutants of dihydrofolate reductase | journal = Science | volume = 239 | issue = 4844 | pages = 1105–10 | date = March 1988 | pmid = 3125607 | doi = 10.1126/science.3125607 | bibcode = 1988Sci...239.1105B }}</ref>

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 ====
[[File:Structural alignment of ba sa ec sp dhfr.png|thumb|class=skin-invert-image|[[Structural alignment]] of chromosomal (Type I) dihydrofolate reductase from ''Bacillus anthracis'' (BaDHFR), ''Staphylococcus aureus'' (SaDHFR), ''Escherichia coli'' (EcDHFR), and ''Streptococcus pneumoniae'' (SpDHFR)]]

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.<ref name="pmid20882962">{{cite journal | vauthors = Beierlein JM, Karri NG, Anderson AC | title = Targeted mutations of Bacillus anthracis dihydrofolate reductase condense complex structure−activity relationships | journal = Journal of Medicinal Chemistry | volume = 53 | issue = 20 | pages = 7327–36 | date = October 2010 | pmid = 20882962 | pmc = 3618964 | doi = 10.1021/jm100727t }}</ref> Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.<ref name="pmid20882962"/>

== As a research tool ==

DHFR has been used as a tool to detect [[protein–protein interactions]] in a [[protein-fragment complementation assay]] (PCA), using a split-protein approach.<ref name="Tarrasov2008">{{cite journal | vauthors = Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, Shames I, Malitskaya Y, Vogel J, Bussey H, Michnick SW | title = An in vivo map of the yeast protein interactome | journal = Science | volume = 320 | issue = 5882 | pages = 1465–70 | date = June 2008 | pmid = 18467557 | doi = 10.1126/science.1153878 | bibcode = 2008Sci...320.1465T | s2cid = 1732896 | url = http://www-nmr.cabm.rutgers.edu/academics/biochem694/reading/Tarassov_et_al_2008.pdf }}</ref>

DHFR-lacking [[Chinese hamster ovary cell|CHO cells]] are the most commonly used [[cell line]] for the production of recombinant proteins. These cells are [[transfection|transfected]] with a [[plasmid]] carrying the ''dhfr'' gene and the gene for the recombinant protein in a single [[expression system]], and then subjected to [[selection (biology)|selective conditions]] in thymidine-lacking [[growth medium|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.<ref>{{cite book | vauthors = Ng SK |chapter=Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker |title=Protein Expression in Mammalian Cells |series=Methods in Molecular Biology |date=2012 |volume=801 |pages=161–172 |doi=10.1007/978-1-61779-352-3_11 |pmid=21987253|isbn=978-1-61779-351-6 }}</ref>

== Interactions ==

Dihydrofolate reductase has been shown to interact with [[GroEL]]<ref name="pmid8559246">{{cite journal | vauthors = Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P, Hartl FU | title = Protein folding in the central cavity of the GroEL-GroES chaperonin complex | journal = Nature | volume = 379 | issue = 6564 | pages = 420–6 | date = February 1996 | pmid = 8559246 | doi = 10.1038/379420a0 | bibcode = 1996Natur.379..420M | s2cid = 4310511 }}</ref> and [[Mdm2]].<ref name="pmid18451149">{{cite journal | vauthors = Maguire M, Nield PC, Devling T, Jenkins RE, Park BK, Polański R, Vlatković N, Boyd MT | title = MDM2 regulates dihydrofolate reductase activity through monoubiquitination | journal = Cancer Research | volume = 68 | issue = 9 | pages = 3232–42 | date = May 2008 | pmid = 18451149 | pmc = 3536468 | doi = 10.1158/0008-5472.CAN-07-5271 }}</ref>

== Interactive pathway map ==
{{FluoropyrimidineActivity WP1601|highlight=Dihydrofolate_reductase}}

== References ==
{{reflist|33em}}

== Further reading ==
{{refbegin | colwidth=33em}}
* {{cite journal | vauthors = Joska TM, Anderson AC | title = Structure-activity relationships of Bacillus cereus and Bacillus anthracis dihydrofolate reductase: toward the identification of new potent drug leads | journal = Antimicrobial Agents and Chemotherapy | volume = 50 | issue = 10 | pages = 3435–43 | date = October 2006 | pmid = 17005826 | pmc = 1610094 | doi = 10.1128/AAC.00386-06 }}
* {{cite journal | vauthors = Chan DC, Fu H, Forsch RA, Queener SF, Rosowsky A | title = 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 = Journal of Medicinal Chemistry | volume = 48 | issue = 13 | pages = 4420–31 | date = June 2005 | pmid = 15974594 | doi = 10.1021/jm0581718 }}
* {{cite journal | vauthors = Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick R, Bertino JR | title = Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase | journal = Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease | volume = 1587 | issue = 2–3 | pages = 164–73 | date = July 2002 | pmid = 12084458 | doi = 10.1016/S0925-4439(02)00079-0 | doi-access = free }}
* {{cite journal | vauthors = Stockman BJ, Nirmala NR, Wagner G, Delcamp TJ, DeYarman MT, Freisheim JH | title = Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution | journal = Biochemistry | volume = 31 | issue = 1 | pages = 218–29 | date = January 1992 | pmid = 1731871 | doi = 10.1021/bi00116a031 }}
* {{cite journal | vauthors = Beltzer JP, Spiess M | title = In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles | journal = The EMBO Journal | volume = 10 | issue = 12 | pages = 3735–42 | date = December 1991 | pmid = 1935897 | pmc = 453108 | doi =  10.1002/j.1460-2075.1991.tb04942.x}}
* {{cite journal | vauthors = Davies JF, Delcamp TJ, Prendergast NJ, Ashford VA, Freisheim JH, Kraut J | title = Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate | journal = Biochemistry | volume = 29 | issue = 40 | pages = 9467–79 | date = October 1990 | pmid = 2248959 | doi = 10.1021/bi00492a021 }}
* {{cite journal | vauthors = Will CL, Dolnick BJ | title = 5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells | journal = The Journal of Biological Chemistry | volume = 264 | issue = 35 | pages = 21413–21 | date = December 1989 | doi = 10.1016/S0021-9258(19)30096-1 | pmid = 2592384 | doi-access = free }}
* {{cite journal | vauthors = Masters JN, Attardi G | title = Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site | journal = Molecular and Cellular Biology | volume = 5 | issue = 3 | pages = 493–500 | date = March 1985 | pmid = 2859520 | pmc = 366741 | doi =  10.1128/mcb.5.3.493}}
* {{cite journal | vauthors = Miszta H, Dabrowski Z, Lanotte M | title = In vitro patterns of enzymic tetrahydrofolate dehydrogenase (EC 1.5.1.3) expression in bone marrow stromal cells | journal = Leukemia | volume = 2 | issue = 11 | pages = 754–9 | date = November 1988 | pmid = 3185016 }}
* {{cite journal | vauthors = Oefner C, D'Arcy A, Winkler FK | title = Crystal structure of human dihydrofolate reductase complexed with folate | journal = European Journal of Biochemistry | volume = 174 | issue = 2 | pages = 377–85 | date = June 1988 | pmid = 3383852 | doi = 10.1111/j.1432-1033.1988.tb14108.x | doi-access = free }}
* {{cite journal | vauthors = Yang JK, Masters JN, Attardi G | title = Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes | journal = Journal of Molecular Biology | volume = 176 | issue = 2 | pages = 169–87 | date = June 1984 | pmid = 6235374 | doi = 10.1016/0022-2836(84)90419-4 }}
* {{cite journal | vauthors = Masters JN, Yang JK, Cellini A, Attardi G | title = A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA | journal = Journal of Molecular Biology | volume = 167 | issue = 1 | pages = 23–36 | date = June 1983 | pmid = 6306253 | doi = 10.1016/S0022-2836(83)80032-1 }}
* {{cite journal | vauthors = Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW | title = The functional human dihydrofolate reductase gene | journal = The Journal of Biological Chemistry | volume = 259 | issue = 6 | pages = 3933–43 | date = March 1984 | doi = 10.1016/S0021-9258(17)43186-3 | pmid = 6323448 | doi-access = free }}
* {{cite journal | vauthors = Funanage VL, Myoda TT, Moses PA, Cowell HR | title = Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5 | journal = Molecular and Cellular Biology | volume = 4 | issue = 10 | pages = 2010–6 | date = October 1984 | pmid = 6504041 | pmc = 369017 | doi =  10.1128/mcb.4.10.2010}}
* {{cite journal | vauthors = Masters JN, Attardi G | title = The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase | journal = Gene | volume = 21 | issue = 1–2 | pages = 59–63 | year = 1983 | pmid = 6687716 | doi = 10.1016/0378-1119(83)90147-6 }}
* {{cite journal | vauthors = Morandi C, Masters JN, Mottes M, Attardi G | title = Multiple forms of human dihydrofolate reductase messenger RNA. Cloning and expression in ''Escherichia coli'' of their DNA coding sequence | journal = Journal of Molecular Biology | volume = 156 | issue = 3 | pages = 583–607 | date = April 1982 | pmid = 6750132 | doi = 10.1016/0022-2836(82)90268-6 }}
* {{cite journal | vauthors = Bonifaci N, Sitia R, Rubartelli A | title = Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding | journal = AIDS | volume = 9 | issue = 9 | pages = 995–1000 | date = September 1995 | pmid = 8527095 | doi = 10.1097/00002030-199509000-00003 | s2cid = 8417864 }}
* {{cite journal | vauthors = Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P, Hartl FU | title = Protein folding in the central cavity of the GroEL-GroES chaperonin complex | journal = Nature | volume = 379 | issue = 6564 | pages = 420–6 | date = February 1996 | pmid = 8559246 | doi = 10.1038/379420a0 | bibcode = 1996Natur.379..420M | s2cid = 4310511 }}
* {{cite journal | vauthors = Gross M, Robinson CV, Mayhew M, Hartl FU, Radford SE | title = Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling | journal = Protein Science | volume = 5 | issue = 12 | pages = 2506–13 | date = December 1996 | pmid = 8976559 | pmc = 2143321 | doi = 10.1002/pro.5560051213 }}
* {{cite journal | vauthors = Schleiff E, Shore GC, Goping IS | title = Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins | journal = FEBS Letters | volume = 404 | issue = 2–3 | pages = 314–8 | date = March 1997 | pmid = 9119086 | doi = 10.1016/S0014-5793(97)00145-2 | s2cid = 29177508 | doi-access = free | bibcode = 1997FEBSL.404..314S }}
* {{cite journal | vauthors = Cody V, Galitsky N, Luft JR, Pangborn W, Rosowsky A, Blakley RL | title = 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 | journal = Biochemistry | volume = 36 | issue = 45 | pages = 13897–903 | date = November 1997 | pmid = 9374868 | doi = 10.1021/bi971711l }}
* {{cite journal | vauthors = Vanguri VK, Wang S, Godyna S, Ranganathan S, Liau G | title = Thrombospondin-1 binds to polyhistidine with high affinity and specificity | journal = The Biochemical Journal | volume = 347 | issue = Pt 2 | pages = 469–73 | date = April 2000 | pmid = 10749676 | pmc = 1220979 | doi = 10.1042/0264-6021:3470469 }}
{{refend}}

== External links ==
* [http://www.nobel.se/medicine/laureates/1988/hitchings-lecture.pdf 1988 Nobel lecture in Medicine]
* [https://proteopedia.org/wiki/index.php/Dihydrofolate_reductase Proteopedia: ''Dihydrofolate reductase'']
* {{PDBe-KB2|P00374|Dihydrofolate reductase}}

{{PDB Gallery|geneid=1719}}
{{Metabolism of vitamins, coenzymes, and cofactors}}
{{Nucleotide metabolism enzymes}}
{{CH-NH oxidoreductases}}
{{Enzymes}}
{{Portal bar|Biology|border=no}}

{{InterPro content|IPR001796}}

{{InterPro content|IPR009159}}

{{DEFAULTSORT:Dihydrofolate Reductase}}
[[Category:Protein domains]]
[[Category:EC 1.5.1]]
[[Category:Enzymes of known structure]]