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Dihydrofolate reductase

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Title: Dihydrofolate reductase  
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Subject: Enzyme kinetics, Enzyme, HAT medium, Chinese hamster ovary cell, Purine metabolism
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Dihydrofolate reductase

Dihydrofolate reductase
Crystal structure of chicken liver dihydrofolate reductase. PDB entry
EC number
CAS number 9002-03-3
IntEnz IntEnz view
ExPASy NiceZyme view
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Dihydrofolate reductase
Symbol DHFR_1
Pfam PF00186
Pfam clan CL0387
InterPro IPR001796
SCOP 1dhi
R67 dihydrofolate reductase
High-resolution structure of a plasmid-encoded dihydrofolate reductase from E.coli. PDB entry
Symbol DHFR_2
Pfam PF06442
InterPro IPR009159
SCOP 1vif
Dihydrofolate reductase
Ribbon diagram of human dihydrofolate reductase in complex with folate (blue). From ​.
Available structures
PDB Ortholog search: PDBe, RCSB
Symbols  ; DHFRP1; DYR
External IDs ChEMBL: GeneCards:
EC number
Species Human Mouse
RefSeq (mRNA)
RefSeq (protein)
Location (UCSC)
PubMed search

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 DHFR gene.[1][2] It is found in the q11→q22 region of chromosome 5.[3] Bacterial species possesses distinct DHFR enzymes (based on their pattern of binding diaminoheterocyclic molecules), but mammalian DHFRs are highly similar.[4]


  • Structure 1
  • Function 2
  • Mechanism 3
  • Clinical significance 4
  • Therapeutic applications 5
    • Potential anthrax treatment 5.1
  • As a research tool 6
  • Interactions 7
  • Interactive pathway map 8
  • References 9
  • Further reading 10
  • External links 11


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 conserved Pro-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 


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]


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 reduction of dihydrofolate to tetrahydrofolate. 

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.[13]

Therapeutic applications

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.[14] Other drugs include trimethoprim and pyrimethamine. These three are widely used as antitumor and antimicrobial agents.[15] Whether or not these are potent anticancer agents is unclear.

Trimethoprim has shown to have activity against a variety of Gram-positive bacterial pathogens.[16] 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.[17][18][19] 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.[16]

Folic acid is necessary for growth,[20] 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.[21] Further studies into inhibitors of DHFR can lead to more ways to treat cancer.

Potential anthrax treatment

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.

Structural alignment of dihydrofolate reductase from Bacillus anthracis (BaDHFR), Staphylococcus aureus (SaDHFR), Escherichia coli (EcDHFR), and Streptococcus pneumoniae (SpDHFR). 

BaDHFR's resistance to trimethoprim analogs is due to these two residues (F96 and Y102), which also confer improved kinetics and catalytic efficiency.[22] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.[22]

As a research tool

DHFR has been used as a tool to detect protein-protein interactions in a protein-fragment complementation assay (PCA).


Dihydrofolate reductase has been shown to interact with GroEL[23] and Mdm2.[24]

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles. [§ 1]

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Fluorouracil (5-FU) Activity edit
  1. ^ The interactive pathway map can be edited at WikiPathways: "FluoropyrimidineActivity_WP1601". 


  1. ^ Chen MJ, Shimada T, Moulton AD, Harrison M, Nienhuis AW (December 1982). "Intronless human dihydrofolate reductase genes are derived from processed RNA molecules". Proc. Natl. Acad. Sci. U.S.A. 79 (23): 7435–9.  
  2. ^ Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW (March 1984). "The functional human dihydrofolate reductase gene". J. Biol. Chem. 259 (6): 3933–43.  
  3. ^ Funanage VL, Myoda TT, Moses PA, Cowell HR (October 1984). "Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5". Mol. Cell. Biol. 4 (10): 2010–6.  
  4. ^ Smith SL, Patrick P, Stone D, Phillips AW, Burchall JJ (November 1979). "Porcine liver dihydrofolate reductase. Purification, properties, and amino acid sequence". J. Biol. Chem. 254 (22): 11475–84.  
  5. ^ 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.  
  6. ^ Filman DJ, Bolin JT, Matthews DA, Kraut J (November 1982). "Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis". J. Biol. Chem. 257 (22): 13663–72.  
  7. ^ a b Osborne 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.  
  8. ^ Bolin JT, Filman DJ, Matthews DA, Hamlin RC, Kraut J (November 1982). "Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate". J. Biol. Chem. 257 (22): 13650–62.  
  9. ^ a b "Entrez Gene: DHFR dihydrofolate reductase". 
  10. ^ a b Schnell JR, Dyson HJ, Wright PE (2004). "Structure, dynamics, and catalytic function of dihydrofolate reductase". Annu Rev Biophys Biomol Struct 33 (1): 119–40.  
  11. ^ Urlaub G, Chasin LA (July 1980). "Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity". Proc. Natl. Acad. Sci. U.S.A. 77 (7): 4216–20.  
  12. ^ Crabtree MJ, Tatham AL, Hale AB, Alp NJ, Channon KM (2009). "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". J. Biol. Chem. 284 (41): 28128–36.  
  13. ^ Cowman AF, Lew AM (November 1989). "Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi". Mol. Cell. Biol. 9 (11): 5182–8.  
  14. ^ 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". J. Mol. Biol. 295 (2): 307–23.  
  15. ^ Benkovic SJ, Fierke CA, Naylor AM (March 1988). "Insights into enzyme function from studies on mutants of dihydrofolate reductase". Science 239 (4844): 1105–10.  
  16. ^ a b Hawser S, Lociuro S, Islam K (March 2006). "Dihydrofolate reductase inhibitors as antibacterial agents". Biochem. Pharmacol. 71 (7): 941–8.  
  17. ^ 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". Nat. Struct. Biol. 2 (11): 1018–25.  
  18. ^ Huennekens FM (June 1996). "In search of dihydrofolate reductase". Protein Sci. 5 (6): 1201–8.  
  19. ^ Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick R, Bertino JR (July 2002). "Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase". Biochim. Biophys. Acta 1587 (2-3): 164–73.  
  20. ^ Bailey SW, Ayling JE (2009). "The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake". Proc. Natl. Acad. Sci. U.S.A. 106 (36): 15424–9.  
  21. ^ Murad AM, Santiago FF, Petroianu A, Rocha PR, Rodrigues MA, Rausch M (July 1993). "Modified therapy with 5-fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer". Cancer 72 (1): 37–41.  
  22. ^ a b Beierlein JM, Karri NG, Anderson AC (October 2010). "Targeted mutations of Bacillus anthracis dihydrofolate reductase condense complex structure−activity relationships". J. Med. Chem. 53 (20): 7327–36.  
  23. ^ 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.  
  24. ^ Maguire M, Nield PC, Devling T, Jenkins RE, Park BK, Polański R, Vlatković N, Boyd MT (May 2008). "MDM2 regulates dihydrofolate reductase activity through monoubiquitination". Cancer Res. 68 (9): 3232–42.  

Further reading

  • Joska TM, Anderson AC (October 2006). "Structure-activity relationships of Bacillus cereus and Bacillus anthracis dihydrofolate reductase: toward the identification of new potent drug leads". Antimicrob. Agents Chemother. 50 (10): 3435–43.  
  • 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". J. Med. Chem. 48 (13): 4420–31.  
  • Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick R, Bertino JR (2002). "Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase". Biochim. Biophys. Acta 1587 (2-3): 164–73.  
  • Stockman BJ, Nirmala NR, Wagner G, Delcamp TJ, DeYarman MT, Freisheim JH (1992). "Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution". Biochemistry 31 (1): 218–29.  
  • Beltzer JP, Spiess M (1991). "In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles". EMBO J. 10 (12): 3735–42.  
  • Davies JF, Delcamp TJ, Prendergast NJ, Ashford VA, Freisheim JH, Kraut J (1990). "Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate". Biochemistry 29 (40): 9467–79.  
  • Will CL, Dolnick BJ (1989). "5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells". J. Biol. Chem. 264 (35): 21413–21.  
  • Masters JN, Attardi G (1985). "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". Mol. Cell. Biol. 5 (3): 493–500.  
  • Miszta H, Dabrowski Z, Lanotte M (1988). "In vitro patterns of enzymic tetrahydrofolate dehydrogenase (EC expression in bone marrow stromal cells". Leukemia 2 (11): 754–9.  
  • Oefner C, D'Arcy A, Winkler FK (1988). "Crystal structure of human dihydrofolate reductase complexed with folate". Eur. J. Biochem. 174 (2): 377–85.  
  • Yang JK, Masters JN, Attardi G (1984). "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". J. Mol. Biol. 176 (2): 169–87.  
  • Masters JN, Yang JK, Cellini A, Attardi G (1983). "A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA". J. Mol. Biol. 167 (1): 23–36.  
  • Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW (1984). "The functional human dihydrofolate reductase gene". J. Biol. Chem. 259 (6): 3933–43.  
  • Funanage VL, Myoda TT, Moses PA, Cowell HR (1984). "Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5". Mol. Cell. Biol. 4 (10): 2010–6.  
  • Masters JN, Attardi G (1983). "The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase". Gene 21 (1-2): 59–63.  
  • Morandi C, Masters JN, Mottes M, Attardi G (1982). "Multiple forms of human dihydrofolate reductase messenger RNA. Cloning and expression in Escherichia coli of their DNA coding sequence". J. Mol. Biol. 156 (3): 583–607.  
  • Bonifaci N, Sitia R, Rubartelli A (1995). "Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding". AIDS 9 (9): 995–1000.  
  • Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P, Hartl FU (1996). "Protein folding in the central cavity of the GroEL-GroES chaperonin complex". Nature 379 (6564): 420–6.  
  • Gross M, Robinson CV, Mayhew M, Hartl FU, Radford SE (1996). "Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling". Protein Sci. 5 (12): 2506–13.  
  • Schleiff E, Shore GC, Goping IS (1997). "Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins". FEBS Lett. 404 (2-3): 314–8.  
  • Cody V, Galitsky N, Luft JR, Pangborn W, Rosowsky A, Blakley RL (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.  
  • Vanguri VK, Wang S, Godyna S, Ranganathan S, Liau G (2000). "Thrombospondin-1 binds to polyhistidine with high affinity and specificity". Biochem. J. 347 (Pt 2): 469–73.  

External links

  • 1988 Nobel lecture in Medicine
  • Dihydrofolate reductaseProteopedia:

This article incorporates text from the public domain Pfam and InterPro IPR001796

This article incorporates text from the public domain Pfam and InterPro IPR009159

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