Dihydrofolate reductase

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Dihydrofolate reductase
PDB 8dfr EBI.jpg
Crystal structure of chicken liver dihydrofolate reductase. PDB entry 8dfr
Identifiers
EC number 1.5.1.3
CAS number 9002-03-3
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Dihydrofolate reductase
Identifiers
Symbol DHFR_1
Pfam PF00186
Pfam clan CL0387
InterPro IPR001796
PROSITE PDOC00072
SCOP 1dhi
SUPERFAMILY 1dhi
R67 dihydrofolate reductase
PDB 2gqv EBI.jpg
High-resolution structure of a plasmid-encoded dihydrofolate reductase from E.coli. PDB entry 2gqv
Identifiers
Symbol DHFR_2
Pfam PF06442
InterPro IPR009159
SCOP 1vif
SUPERFAMILY 1vif
Dihydrofolate reductase
Dihydrofolate reductase 1DRF.png
Ribbon diagram of human dihydrofolate reductase in complex with folate (blue). From PDB 1DRF.
Available structures
PDB Ortholog search: PDBe, RCSB
Identifiers
Symbols DHFR ; DHFRP1; DYR
External IDs OMIM126060 MGI94890 HomoloGene56470 ChEMBL: 202 GeneCards: DHFR Gene
EC number 1.5.1.3
Orthologs
Species Human Mouse
Entrez 1719 13361
Ensembl ENSG00000228716 ENSMUSG00000021707
UniProt P00374 P00375
RefSeq (mRNA) NM_000791 NM_010049
RefSeq (protein) NP_000782 NP_034179
Location (UCSC) Chr 5:
79.92 – 79.95 Mb
Chr 13:
92.35 – 92.39 Mb
PubMed search [1] [2]

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[edit]

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 

Function[edit]

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. 

Mechanism[edit]

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. 

Biological function[edit]

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]

Clinical significance[edit]

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. Its central role in DNA precursor synthesis, coupled with its inhibition by antagonists such as trimethoprim and methotrexate, which are used as anti-bacterial or anti-cancer agents, has made DHFR a target of anticancer chemotherapy. However, resistance has developed against some drugs, as a result of changes in DHFR itself.[13]

Therapeutic application and disease relevance[edit]

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.

Dihydrofolate reductase as a target in the treatment of anthrax[edit]

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]

Application as a research tool[edit]

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

Interactions[edit]

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

Interactive pathway map[edit]

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

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

References[edit]

  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. doi:10.1073/pnas.79.23.7435. PMC 347354. PMID 6961421. 
  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. PMID 6323448. 
  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. PMC 369017. PMID 6504041. 
  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. PMID 500653. 
  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): 452–455. doi:10.1126/science.17920. PMID 17920. 
  6. ^ Filman DJ, Bolin JT, Matthews DA, Kraut J. (November 1982). "Crystal structure of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis". The Journal of Biological Chemistry 257 (22): 13650–13662. PMID 6815179. 
  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. doi:10.1021/bi010621k. PMID 11502178. 
  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. PMID 6815178. 
  9. ^ a b "Entrez Gene: DHFR dihydrofolate reductase". 
  10. ^ a b Schnell JR, Dyson HJ, Wright PE (June 2004). "Structure, dynamics, and catalytic function of dihydrofolate reductase.". Annual Review of Biophysics and Biomolecular Structure 33 (1): 119–40. doi:10.1146/annurev.biophys.33.110502.133613. PMID 15139807. 
  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. doi:10.1073/pnas.77.7.4216. PMC 349802. PMID 6933469. 
  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". Journal of Biological Chemistry 284 (41): 28128–36. doi:10.1074/jbc.M109.041483. PMC 2788863. PMID 19666465. 
  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. PMC 363670. PMID 2601715. 
  14. ^ Li R, Sirawaraporn R, Chitnumsub P, et al. (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. doi:10.1006/jmbi.1999.3328. PMID 10623528. 
  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. doi:10.1126/science.3125607. PMID 3125607. 
  16. ^ a b Hawser S, Lociuro S, Islam K (March 2006). "Dihydrofolate reductase inhibitors as antibacterial agents". Biochem. Pharmacol. 71 (7): 941–8. doi:10.1016/j.bcp.2005.10.052. PMID 16359642. 
  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. doi:10.1038/nsb1195-1018. PMID 7583655. 
  18. ^ Huennekens FM (June 1996). "In search of dihydrofolate reductase". Protein Sci. 5 (6): 1201–8. doi:10.1002/pro.5560050626. PMC 2143423. PMID 8762155. 
  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. doi:10.1016/S0925-4439(02)00079-0. PMID 12084458. 
  20. ^ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730961/
  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. doi:10.1002/1097-0142(19930701)72:1<37::AID-CNCR2820720109>3.0.CO;2-P. PMID 8508427. 
  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. doi:10.1021/jm100727t. PMC 3618964. PMID 20882962. 
  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. doi:10.1038/379420a0. PMID 8559246. 
  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. doi:10.1158/0008-5472.CAN-07-5271. PMC 3536468. PMID 18451149. 

Further reading[edit]

External links[edit]

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

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