Dihydrofolate reductase

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The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)
The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)

The enzyme dihydrofolate reductase (DHFR) occurs in all organisms and has been particularly well-studied in the bacterium Escherichia coli and in humans[1][2][3]. It catalyzes the reduction of dihydrofolate to tetrahydrofolate, with NADPH acting as hydride donor. The human enzyme is a target for developing inhibitors used in anti-cancer chemotherapies[4], while the bacterial enzymes are targets for developing inhibitors as antibiotics. DHFR is a model enzyme for studying the kinetics, mechanism, and inhibition of enzymatic reactions and the underlying structure and conformational dynamics.


DHFR occurs in all organisms and most cells

E. coli (left) and human (right) DHFR have a similar architecture and mode of binding to NADPH(green) and the competitive inhibitor methotrexate(purple). Original image by David Goodsell
E. coli (left) and human (right) DHFR have a similar architecture and mode of binding to NADPH(green) and the competitive inhibitor methotrexate(purple). Original image by David Goodsell

DHFR is found in all organisms. Some bacteria acquire resistance to DHFR inhibitors through expressing a second form of DHFR coded on a plasmid. The enzymes from E. coli (ecDHFR) and humans (hDHFR) have similar folds, while the plasmid-encoded enzyme has an unrelated fold. In humans, DHFR is expressed in most tissues[1], and there are two genes, DHFR and DHFR2/DHFRL1, the latter targeted to mitochondria[5]. Mice and rats lack the second gene but also show DHFR activity in mitochondria[6].

Reactions catalyzed

Dihydrofolate reductase (DHFR, [2]) is an enzyme which uses the co-factor NADPH as electron donor. It catalyzes the reduction of as NADPH is oxidized to NADP+. The mammalian enzymes also accept folic acid as a substrate, reducing it to THF. This allows the use of folic acid, which is easier to synthesize than DHF or THF, to fortify food.[7][8]. Some bacterial enzymes also accept folic acid as a substrate [9] but it acts as a competitive inhibitor in the E. coli enzyme.

The folate is a form of the essential vitamin B9. Folate is not part of our natural diet (it contains dihydrofolate and tetrahydrofolate, sometimes as a poly-glutamate conjugate) but is bioavailable and simpler to synthesize.


Tetrahydrofolate is an essential cofactor of one-carbon metabolism[10][11] (for a visual, see here). For example, it is required for turning homocysteine into the amino acid methionine, and for biosynthesis of dTTP, one of the four nucleotide building blocks of DNA, from dUTP. In these reactions, tetrahydrofolate is first methylated and then oxidized to dihydrofolate. To allow for multiple rounds of turnover, dihydrofolate has to be reduced again; dihydrofolate reductase is the enzyme that enables this. Like many common cofactors, tetrahydrofolate is not synthesized de novo in the human body. Instead, it is provided as vitamin B9 in a healthy diet, for instance through leafy vegetables (the name for folic acid come from e.g. Latin folio, leaf). Many countries fortify common food ingredients like flour with vitamin B9 (in the form of folic acid) to ensure sufficient dietary levels. As described below, DHFR also plays a role in reducing folic acid to the biologically active tetrahydrofolate.

Structure and Function

See also

3D Structures of Dihydrofolate reductase

Dihydrofolate reductase 3D structures

Additional Resources


  1. Schnell JR, Dyson HJ, Wright PE. Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu Rev Biophys Biomol Struct. 2004;33:119-40. doi:, 10.1146/annurev.biophys.33.110502.133613. PMID:15139807 doi:http://dx.doi.org/10.1146/annurev.biophys.33.110502.133613
  2. https://en.wikipedia.org/wiki/Dihydrofolate_reductase
  3. https://pdb101.rcsb.org/motm/34
  4. Raimondi MV, Randazzo O, La Franca M, Barone G, Vignoni E, Rossi D, Collina S. DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents. Molecules. 2019 Mar 22;24(6). pii: molecules24061140. doi:, 10.3390/molecules24061140. PMID:30909399 doi:http://dx.doi.org/10.3390/molecules24061140
  5. McEntee G, Minguzzi S, O'Brien K, Ben Larbi N, Loscher C, O'Fagain C, Parle-McDermott A. The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15157-62. doi:, 10.1073/pnas.1103605108. Epub 2011 Aug 26. PMID:21876184 doi:http://dx.doi.org/10.1073/pnas.1103605108
  6. Hughes L, Carton R, Minguzzi S, McEntee G, Deinum EE, O'Connell MJ, Parle-McDermott A. An active second dihydrofolate reductase enzyme is not a feature of rat and mouse, but they do have activity in their mitochondria. FEBS Lett. 2015 Jul 8;589(15):1855-62. doi: 10.1016/j.febslet.2015.05.017. Epub, 2015 May 14. PMID:25980602 doi:http://dx.doi.org/10.1016/j.febslet.2015.05.017
  7. Choi JH, Yates Z, Veysey M, Heo YR, Lucock M. Contemporary issues surrounding folic Acid fortification initiatives. Prev Nutr Food Sci. 2014 Dec;19(4):247-60. doi: 10.3746/pnf.2014.19.4.247. Epub, 2014 Dec 31. PMID:25580388 doi:http://dx.doi.org/10.3746/pnf.2014.19.4.247
  8. https://ods.od.nih.gov/factsheets/Folate-HealthProfessional/
  9. Loveridge EJ, Hroch L, Hughes RL, Williams T, Davies RL, Angelastro A, Luk LY, Maglia G, Allemann RK. Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima. Biochemistry. 2017 Apr 4;56(13):1879-1886. doi: 10.1021/acs.biochem.6b01268. Epub, 2017 Mar 24. PMID:28319664 doi:http://dx.doi.org/10.1021/acs.biochem.6b01268
  10. Fox JT, Stover PJ. Folate-mediated one-carbon metabolism. Vitam Horm. 2008;79:1-44. doi: 10.1016/S0083-6729(08)00401-9. PMID:18804690 doi:http://dx.doi.org/10.1016/S0083-6729(08)00401-9
  11. doi: https://dx.doi.org/10.1007/978-1-4020-2400-9_12
  12. Ivanetich KM, Santi DV. Thymidylate synthase-dihydrofolate reductase in protozoa. Exp Parasitol. 1990 Apr;70(3):367-71. PMID:2178951
  13. Liu CT, Francis K, Layfield JP, Huang X, Hammes-Schiffer S, Kohen A, Benkovic SJ. Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100. Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):18231-6. doi:, 10.1073/pnas.1415940111. Epub 2014 Dec 1. PMID:25453098 doi:http://dx.doi.org/10.1073/pnas.1415940111
  14. Sawaya MR, Kraut J. Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry. 1997 Jan 21;36(3):586-603. PMID:9012674 doi:http://dx.doi.org/10.1021/bi962337c
  15. Wan Q, Bennett BC, Wilson MA, Kovalevsky A, Langan P, Howell EE, Dealwis C. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. Proc Natl Acad Sci U S A. 2014 Dec 1. pii: 201415856. PMID:25453083 doi:http://dx.doi.org/10.1073/pnas.1415856111
  16. Stojkovic V, Perissinotti LL, Willmer D, Benkovic SJ, Kohen A. Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction. J Am Chem Soc. 2012 Jan 25;134(3):1738-45. doi: 10.1021/ja209425w. Epub 2012 Jan , 17. PMID:22171795 doi:http://dx.doi.org/10.1021/ja209425w
  17. doi: https://dx.doi.org/10.3390/quantum3010006
  18. Wrobel A, Arciszewska K, Maliszewski D, Drozdowska D. Trimethoprim and other nonclassical antifolates an excellent template for searching modifications of dihydrofolate reductase enzyme inhibitors. J Antibiot (Tokyo). 2020 Jan;73(1):5-27. doi: 10.1038/s41429-019-0240-6. Epub, 2019 Oct 2. PMID:31578455 doi:http://dx.doi.org/10.1038/s41429-019-0240-6

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