Dihydrofolate reductase

Dihydrofolate reductase
Crystal structure of chicken liver dihydrofolate reductase. PDB entry 8dfr
Identifiers
EC number	1.5.1.3
CAS number	Template:CAS
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
Search PMC articles PubMed articles NCBI proteins

Dihydrofolate reductase
Identifiers
Symbol	DHFR_1
Pfam	PF00186
Pfam clan	CL0387
InterPro	IPR001796
PROSITE	PDOC00072
SCOP	1dhi
SUPERFAMILY	1dhi
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

R67 dihydrofolate reductase
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
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Dihydrofolate reductase
Ribbon diagram of human dihydrofolate reductase in complex with folate (blue). From PDB: 1DRF​.
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
Identifiers
Symbols	DHFR ; DHFRP1; DYR
External IDs	OMIM: 126060 MGI: 94890 HomoloGene: 56470 ChEMBL: 202 GeneCards: DHFR Gene
EC number	1.5.1.3
Gene ontology Molecular function • mRNA binding • dihydrofolate reductase activity • drug binding • NADP binding Cellular component • cellular_component • nucleoplasm • cytosol Biological process • G1/S transition of mitotic cell cycle • regulation of transcription involved in G1/S transition of mitotic cell cycle • mitotic cell cycle • glycine biosynthetic process • one-carbon metabolic process • vitamin metabolic process • water-soluble vitamin metabolic process • nucleotide biosynthetic process • response to methotrexate • small molecule metabolic process • nitric oxide metabolic process • tetrahydrofolate metabolic process • tetrahydrofolate biosynthetic process • folic acid metabolic process • regulation of nitric-oxide synthase activity • oxidation-reduction process Sources: Amigo / QuickGO
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: 80.63 – 80.65 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. It is found in the q11→q22 region of chromosome 5. Bacterial species possesses distinct DHFR enzymes (based on their pattern of binding diaminoheterocyclic molecules), but mammalian DHFRs are highly similar.

Structure

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

Human DHFR with bound dihydrofolate and NADPH

Function

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.

Reaction catalyzed by DHFR.  Tetrahydrofolate synthesis pathway. 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. 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. DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin

Mechanism

The reduction of dihydrofolate to tetrahydrofolate.

DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate. 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.

Clinical significance

Dihydrofolate reductase deficiency has been linked to megaloblastic anemia. 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.

Therapeutic applications

Main article: Dihydrofolate reductase inhibitor

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. Other drugs include trimethoprim and pyrimethamine. These three are widely used as antitumor and antimicrobial agents.

Trimethoprim has shown to have activity against a variety of Gram-positive bacterial pathogens. 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. 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.

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

Bacteria also need DHFR to grow and multiply and hence inhibitors selective for bacterial DHFR have found application as antibacterial agents.

Classes of small-molecules employed as inhibitors of dihydrofolate reductase include diaminoquinazoline & diaminopyrroloquinazoline, diaminopyrimidine, diaminopteridine and diaminotriazines.

Potential anthrax treatment

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

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.

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

As a research tool

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

Interactions

Dihydrofolate reductase has been shown to interact with GroEL and Mdm2.

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles.

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|{{{bSize}}}px|alt=Fluorouracil (5-FU) Activity edit]]

File:FluoropyrimidineActivity_WP1601.png

Fluorouracil (5-FU) Activity edit

External links

• 1988 Nobel lecture in Medicine
• Proteopedia: Dihydrofolate reductase

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

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