Dihydrofolate reductase

DHFR
Available structures PDB Ortholog search: PDBe RCSB List of PDB id codes 1BOZ, 1DHF, 1DLR, 1DLS, 1DRF, 1HFP, 1HFQ, 1HFR, 1KMS, 1KMV, 1MVS, 1MVT, 1OHJ, 1OHK, 1PD8, 1PD9, 1PDB, 1S3U, 1S3V, 1S3W, 1U71, 1U72, 1YHO, 2C2S, 2C2T, 2DHF, 2W3A, 2W3B, 2W3M, 3EIG, 3F8Y, 3F8Z, 3F91, 3FS6, 3GHC, 3GHV, 3GHW, 3GI2, 3GYF, 3L3R, 3N0H, 3NTZ, 3NU0, 3NXO, 3NXR, 3NXT, 3NXV, 3NXX, 3NXY, 3NZD, 3OAF, 3S3V, 3S7A, 4DDR, 4G95, 4KAK, 4KBN, 4KD7, 4KEB, 4KFJ, 4M6J, 4M6K, 4M6L, 4QHV, 4QJC
Identifiers
Aliases	DHFR , DHFRP1, DYR, dihydrofolate reductase
External IDs	OMIM: 126060; MGI: 94890; HomoloGene: 56470; GeneCards: DHFR; OMA:DHFR - orthologs
Gene location (Human) Chr. Chromosome 5 (human) [1] Band 5q14.1 Start 80,626,226 bp [1] End 80,654,983 bp [1]
Gene location (Mouse) Chr. Chromosome 13 (mouse) [2] Band 13 C3|13 47.64 cM Start 92,491,234 bp [2] End 92,525,561 bp [2]
RNA expression pattern Bgee Human Mouse (ortholog) Top expressed in buccal mucosa cell ventricular zone ganglionic eminence trabecular bone bone marrow bone marrow cells oocyte inferior ganglion of vagus nerve jejunal mucosa endothelial cell Top expressed in abdominal wall primitive streak human fetus mandibular prominence maxillary prominence epiblast Gonadal ridge efferent ductule Paneth cell somite More reference expression data BioGPS n/a
Gene ontology Molecular function methotrexate binding folic acid binding dihydrofolate reductase activity NADPH binding RNA binding oxidoreductase activity mRNA binding folate reductase activity translation repressor activity, mRNA regulatory element binding sequence-specific mRNA binding NADP binding Cellular component cytosol cytoplasm mitochondrion cellular component Biological process response to methotrexate dihydrofolate metabolic process one-carbon metabolic process axon regeneration regulation of transcription involved in G1/S transition of mitotic cell cycle tetrahydrofolate metabolic process tetrahydrofolate biosynthetic process positive regulation of nitric-oxide synthase activity folic acid metabolic process tetrahydrobiopterin biosynthetic process regulation of removal of superoxide radicals negative regulation of translation Sources:Amigo / QuickGO
Orthologs Species Human Mouse Entrez 1719 13361 Ensembl ENSG00000228716 ENSMUSG00000021707 UniProt P00374 P00375 RefSeq (mRNA) NM_000791 NM_001290354 NM_001290357 NM_010049 RefSeq (protein) NP_000782 NP_001277283 NP_001277286 NP_034179 Location (UCSC) Chr 5: 80.63 – 80.65 Mb Chr 13: 92.49 – 92.53 Mb PubMed search [3] [4]
Wikidata
View/Edit Human View/Edit Mouse

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 cofactors used in one-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the DHFR gene. ^{[5] [6]} It is found in the q14.1 region of chromosome 5. ^[7]

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 mammalian ones remain highly similar. ^[8] The latter (type II), represented by the plastid-encoded R67, is a tiny enzyme that works by forming a homotetramer. ^[9]

Function

Dihydrofolate reductase
Identifiers
EC no.	1.5.1.3
CAS no.	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 / QuickGO
Search PMC articles PubMed articles NCBI proteins

Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a proton 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. ^[10]

• 
  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. ^[11] 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. ^[12] DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin. ^[13]

Structure

Dihydrofolate reductase
Crystal structure of chicken liver dihydrofolate reductase. PDB entry 8dfr
Identifiers
Symbol	DHFR_1
Pfam	PF00186
Pfam clan	CL0387
InterPro	IPR001796
PROSITE	PDOC00072
SCOP2	1dhi / SCOPe / SUPFAM
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

A central eight-stranded beta-pleated sheet makes up the main feature of the polypeptide backbone folding of DHFR. ^[14] Seven of these strands are parallel and the eighth runs antiparallel. Four alpha helices connect successive beta strands. ^[15] 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. ^[16] 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. ^[15]

Mechanism

General mechanism

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. ^[11] 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. ^[16]

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. ^{[17] [18]} In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer. ^[19]

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. ^[19] 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. ^{[17] [20]} 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. ^{[21] [17] [20]} The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation. ^[18] A water molecule is proved to be involved in the protonation step. ^{[22] [23] [24]} Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop. ^[25]

Conformational changes of DHFR

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⁺: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. ^[21]

Conformational changes are critical in DHFR's catalytic mechanism. ^[26] The Met20 loop of DHFR is able to open, close or occlude the active site. ^{[23] [17]} 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. ^[23] 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⁺ by NADPH is prior to product dissociation. Thus, the next round of reaction can occur upon the binding of substrate. ^[21]

R67 DHFR

R67 dihydrofolate reductase
R67 in complex with DHF and NADP+, monomer. PDB entry 2rk1 .
Identifiers
Symbol	DHFR_2
Pfam	PF06442
InterPro	IPR009159
SCOP2	1vif / SCOPe / SUPFAM
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

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. ^[27] 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. ^[28] Compare with E. coli chromosomal DHFR, it has higher K_m 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. ^[29]

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. ^[30]

• 
  Reaction Kinetics comparison between E. coli DHFR (EcDHFR) and R67 DHFR
• 
  Structure difference of substrate binding in EcDHFR and R67 DHFR

Clinical significance

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. ^{[10] [31] [32]}

Therapeutic applications

Main article: 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, resistance has developed against some drugs, as a result of mutational changes in DHFR itself. ^[33]

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. ^[34]

Folate is necessary for growth, ^[35] 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. ^[36] 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. ^[37] Trimethoprim has shown to have activity against a variety of Gram-positive bacterial pathogens. ^[37] 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. ^{[38] [39] [40]} Resistance can arise from DHFR gene amplification, mutations in DHFR, ^{[41] [42]} decrease in the uptake of the drugs, among others. Regardless, trimethoprim and sulfamethoxazole in combination has been used as an antibacterial agent for decades. ^[37]

Pyrimethamine is a widely used antiprotozoal agent. ^[43]

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

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. ^[44] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors. ^[44]

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. ^[45]

DHFR-lacking CHO cells are the most commonly used cell line for the production of recombinant proteins. These cells are transfected with a plasmid carrying the dhfr gene and the gene for the recombinant protein in a single expression system, and then subjected to selective conditions in thymidine-lacking 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. ^[46]

Interactions

Dihydrofolate reductase has been shown to interact with GroEL ^[47] and Mdm2. ^[48]

Interactive pathway map

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

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|alt=Fluorouracil (5-FU) Activity edit]]

Fluorouracil (5-FU) Activity edit

1. The interactive pathway map can be edited at WikiPathways: "FluoropyrimidineActivity_WP1601".

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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". Antimicrobial Agents and Chemotherapy. 50 (10): 3435–43. doi:10.1128/AAC.00386-06. PMC   1610094 . PMID   17005826.
• 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". Journal of Medicinal Chemistry. 48 (13): 4420–31. doi:10.1021/jm0581718. PMID   15974594.
• 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". Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1587 (2–3): 164–73. doi: 10.1016/S0925-4439(02)00079-0 . PMID   12084458.
• Stockman BJ, Nirmala NR, Wagner G, Delcamp TJ, DeYarman MT, Freisheim JH (January 1992). "Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution". Biochemistry. 31 (1): 218–29. doi:10.1021/bi00116a031. PMID   1731871.
• Beltzer JP, Spiess M (December 1991). "In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles". The EMBO Journal. 10 (12): 3735–42. doi:10.1002/j.1460-2075.1991.tb04942.x. PMC   453108 . PMID   1935897.
• Davies JF, Delcamp TJ, Prendergast NJ, Ashford VA, Freisheim JH, Kraut J (October 1990). "Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate". Biochemistry. 29 (40): 9467–79. doi:10.1021/bi00492a021. PMID   2248959.
• Will CL, Dolnick BJ (December 1989). "5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells". The Journal of Biological Chemistry. 264 (35): 21413–21. doi: 10.1016/S0021-9258(19)30096-1 . PMID   2592384.
• Masters JN, Attardi G (March 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". Molecular and Cellular Biology. 5 (3): 493–500. doi:10.1128/mcb.5.3.493. PMC   366741 . PMID   2859520.
• Miszta H, Dabrowski Z, Lanotte M (November 1988). "In vitro patterns of enzymic tetrahydrofolate dehydrogenase (EC 1.5.1.3) expression in bone marrow stromal cells". Leukemia. 2 (11): 754–9. PMID   3185016.
• Oefner C, D'Arcy A, Winkler FK (June 1988). "Crystal structure of human dihydrofolate reductase complexed with folate". European Journal of Biochemistry. 174 (2): 377–85. doi: 10.1111/j.1432-1033.1988.tb14108.x . PMID   3383852.
• Yang JK, Masters JN, Attardi G (June 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". Journal of Molecular Biology. 176 (2): 169–87. doi:10.1016/0022-2836(84)90419-4. PMID   6235374.
• Masters JN, Yang JK, Cellini A, Attardi G (June 1983). "A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA". Journal of Molecular Biology. 167 (1): 23–36. doi:10.1016/S0022-2836(83)80032-1. PMID   6306253.
• Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW (March 1984). "The functional human dihydrofolate reductase gene". The Journal of Biological Chemistry. 259 (6): 3933–43. doi: 10.1016/S0021-9258(17)43186-3 . PMID   6323448.
• 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". Molecular and Cellular Biology. 4 (10): 2010–6. doi:10.1128/mcb.4.10.2010. PMC   369017 . PMID   6504041.
• Masters JN, Attardi G (1983). "The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase". Gene. 21 (1–2): 59–63. doi:10.1016/0378-1119(83)90147-6. PMID   6687716.
• Morandi C, Masters JN, Mottes M, Attardi G (April 1982). "Multiple forms of human dihydrofolate reductase messenger RNA. Cloning and expression in Escherichia coli of their DNA coding sequence". Journal of Molecular Biology. 156 (3): 583–607. doi:10.1016/0022-2836(82)90268-6. PMID   6750132.
• Bonifaci N, Sitia R, Rubartelli A (September 1995). "Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding". AIDS. 9 (9): 995–1000. doi:10.1097/00002030-199509000-00003. PMID   8527095. S2CID   8417864.
• 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. Bibcode:1996Natur.379..420M. doi:10.1038/379420a0. PMID   8559246. S2CID   4310511.
• Gross M, Robinson CV, Mayhew M, Hartl FU, Radford SE (December 1996). "Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling". Protein Science. 5 (12): 2506–13. doi:10.1002/pro.5560051213. PMC   2143321 . PMID   8976559.
• Schleiff E, Shore GC, Goping IS (March 1997). "Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins". FEBS Letters. 404 (2–3): 314–8. doi: 10.1016/S0014-5793(97)00145-2 . PMID   9119086. S2CID   29177508.
• Cody V, Galitsky N, Luft JR, Pangborn W, Rosowsky A, Blakley RL (November 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. doi:10.1021/bi971711l. PMID   9374868.
• Vanguri VK, Wang S, Godyna S, Ranganathan S, Liau G (April 2000). "Thrombospondin-1 binds to polyhistidine with high affinity and specificity". The Biochemical Journal. 347 (Pt 2): 469–73. doi:10.1042/0264-6021:3470469. PMC   1220979 . PMID   10749676.

External links

• 1988 Nobel lecture in Medicine
• Proteopedia: Dihydrofolate reductase
• Overview of all the structural information available in the PDB for UniProt : P00374 (Dihydrofolate reductase) at the PDBe-KB .

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

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