Dihydrofolate reductase

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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.

Contents 1 DHFR occurs in all organisms and most cells 2 Reactions catalyzed 3 Relevance 4 Structure and Function 5 See also 6 3D Structures of Dihydrofolate reductase 7 Acknowledgements 8 References

DHFR occurs in all organisms and most cells

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

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)

Dihydrofolate reductase (DHFR, 1.5.1.3 [2]) is an enzyme which uses the co-factor NADPH as electron donor. It catalyzes the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) 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.

Relevance

Tetrahydrofolate (THF) is an essential cofactor of one-carbon metabolism^[10][11]. For example, THF 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 above, DHFR also plays a role in reducing folic acid to the biologically active tetrahydrofolate.

Structure and Function

Show:Asymmetric Unit Biological Assembly   Export Animated Image Fold and ligand binding DHFR from bacteria and vertebrates share a common fold (CATH, SCOP) featuring a surrounded by . The bind in similar locations in bacterial and vertebrate enzymes. The substrate (and the competive inhibitors ) bind on one side of the beta sheet while the co-factor NADPH wraps around the sheet (around the C-terminal ends of strands as found in the classic Rossmann fold nucleotide binding site), with the nicotinamide moiety next to the substrate and the adenine moiety on the other side of the sheet. In some cases, DHFR forms a complex with the enzymes that produce dihydrofolate, e.g. thymidylate synthase (TS)[12]. Mechanism DHFR is thought to proceed in a multi-step mechanism. Once NADPH and DHF are bound to the E. coli enzyme, DHF is first protonated and then reduced through a hydride transfer from NADPH.[13] Substrate binding and product release is thought to have a definite choreography, with fresh NADPH binding before THF is released. The changes during the catalytic cycle. The enzyme has an occluded conformation where a loop blocks entry of NADPH into the active site, and a closed conformation, where NADPH and DHF are in close contact. A model by M. R. Sawaya and J. Kraut from 1997 based on six isomorphous crystal structures shows the envisioned sequence of events in the movie below [14]. The original web page of this movie is available on the web archive. The was studied using neutron diffraction and high-resolution X-ray diffraction to visualize the hydrogen atoms that are usually not visible in crystal structures.[15] The hydride transfer is thought to involve hydride tunneling, supported by temperature-dependent kinetic isotope effects[16]. Tunneling is a quantum phenomenon explaining how a small particle can cross an activation barrier even when it lacks sufficient activation energy. [17] Inhibitors The human enzyme is sufficiently different from the bacterial enzymes to make specific inhibitors[18]. Inhibitors of the human enzyme are used in cancer treatment while inhibitors of the bacterials enzymes[19] are used as broad-spectrum antibiotics or more specifically to treat a specific infectious disease. An example of in inhibitor of bacterial DHFR is Trimethoprim (TMP). Antifolate inhibitors like (MTX), which is very similar to , are used in cancer therapy, see also Methotrexate. Resistance Antibiotics that inhibit bacterial DHFR are widely used in medicine and agriculture, and some bacterial strains have acquired resistance[20]. For example, some bacteria carry (either on their genome or on a plasmid) the trimethoprim-resistant dfrB gene coding for an enzyme DfrB that has a fold distinct from DHFR (coded by the folA gene)[21]. Like DHFR, Dfrb has dihydrofolate reductase activity, but unlike DHFR, it is not inhibited by trimethoprim. Thus, resistant bacteria can grow even in the presence of trimethoprim, relying on the Dfrb enzyme while the DHFR enzyme is inhibited. The is unique in that the active site has fourfold symmetry, with DHF and NADPH bound by the same amino acid residues from different subunits. In the available crystal structure, two folate molecules are bound (use checkbox to show). The enzyme is non-specific and can bind to unproductive combinations of ligands (two DHF molecules or two NADPH molecules) or to the productive combination of one DHF and one NADPH molecule each.

See also

• An overview of pathways involving folate and S-adenosyl methioine: One-carbon metabolism
• Another page on DHFR: Molecular Playground/DHFR
  
• Fighting malaria with antifolates" Malarial Dihydrofolate Reductase as Drug Target.
  
• Overview of enzymes targeted in cancer therapy: Cancer.

3D Structures of Dihydrofolate reductase

Dihydrofolate reductase 3D structures

Acknowledgements

This page was revised as part of the Spring 2022 Biochemistry II course at WSU. Thanks go to Kia, Anna, Shaylie and Michael for helpful suggestions to improve the page.

References

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
19. ↑ Estrada A, Wright DL, Anderson AC. Antibacterial Antifolates: From Development through Resistance to the Next Generation. Cold Spring Harb Perspect Med. 2016 Aug 1;6(8). pii: cshperspect.a028324. doi:, 10.1101/cshperspect.a028324. PMID:27352799 doi:http://dx.doi.org/10.1101/cshperspect.a028324
20. ↑ Capasso C, Supuran CT. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors. J Enzyme Inhib Med Chem. 2014 Jun;29(3):379-87. doi:, 10.3109/14756366.2013.787422. Epub 2013 Apr 29. PMID:23627736 doi:http://dx.doi.org/10.3109/14756366.2013.787422
21. ↑ Narayana N, Matthews DA, Howell EE, Nguyen-huu X. A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site. Nat Struct Biol. 1995 Nov;2(11):1018-25. PMID:7583655