Biology:Dihydrofolate reductase

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Short description: Mammalian protein found in Homo sapiens


A representation of the 3D structure of the protein myoglobin showing turquoise α-helices.
Generic protein structure example

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 1-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the DHFR gene.[1][2] It is found in the q14.1 region of chromosome 5.[3]

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.[4] The latter (type II), represented by the plastid-encoded R67, is a tiny enzyme that works by forming a homotetramer.[5]

Function

Dihydrofolate reductase
Identifiers
EC number1.5.1.3
CAS number9002-03-3
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO

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.[6]

  • 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.[7] 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.[8] DHFR has also been demonstrated as an enzyme involved in the salvage of tetrahydrobiopterin from dihydrobiopterin[9]


Structure

Dihydrofolate reductase
PDB 8dfr EBI.jpg
Crystal structure of chicken liver dihydrofolate reductase. PDB entry 8dfr
Identifiers
SymbolDHFR_1
PfamPF00186
Pfam clanCL0387
InterProIPR001796
PROSITEPDOC00072
SCOP21dhi / SCOPe / SUPFAM

A central eight-stranded beta-pleated sheet makes up the main feature of the polypeptide backbone folding of DHFR.[10] Seven of these strands are parallel and the eighth runs antiparallel. Four alpha helices connect successive beta strands.[11] 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.[12] 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.[11]

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.[7] 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.[12]

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.[13][14] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.[15]

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.[15] 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.[13][16] 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.[17][13][16] The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.[14] A water molecule is proved to be involved in the protonation step.[18][19][20] Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.[21]

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.[17]

Conformational changes are critical in DHFR's catalytic mechanism.[22] The Met20 loop of DHFR is able to open, close or occlude the active site.[19][13] 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.[19] 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.[17]

R67 DHFR

R67 dihydrofolate reductase
PDB 2rk1 EBI.png
R67 in complex with DHF and NADP+, monomer. PDB entry 2rk1.
Identifiers
SymbolDHFR_2
PfamPF06442
InterProIPR009159
SCOP21vif / SCOPe / SUPFAM

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.[23] 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.[24] Compare with E. coli chromosomal DHFR, it has higher Km 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.[25]

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.[26]

  • 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.[6][27][28]

Therapeutic applications

Main page: Biology: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.[29]

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.[30]

Folate is necessary for growth,[31] 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.[32] 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.[33] Trimethoprim has shown to have activity against a variety of Gram-positive bacterial pathogens.[33] 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.[34][35][36] Resistance can arise from DHFR gene amplification, mutations in DHFR,[37][38] decrease in the uptake of the drugs, among others. Regardless, trimethoprim and sulfamethoxazole in combination has been used as an antibacterial agent for decades.[33]

Pyrimethamine is a widely used antiprotozoal agent.[39]

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.[40] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.[40]

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.[41]

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.[42]

Interactions

Dihydrofolate reductase has been shown to interact with GroEL[43] and Mdm2.[44]

Interactive pathway map

References

  1. ↑ "Intronless human dihydrofolate reductase genes are derived from processed RNA molecules". Proceedings of the National Academy of Sciences of the United States of America 79 (23): 7435–9. December 1982. doi:10.1073/pnas.79.23.7435. PMID 6961421. Bibcode: 1982PNAS...79.7435C. 
  2. ↑ "The functional human dihydrofolate reductase gene". The Journal of Biological Chemistry 259 (6): 3933–43. March 1984. doi:10.1016/S0021-9258(17)43186-3. PMID 6323448. 
  3. ↑ "DHFR dihydrofolate reductase [Homo sapiens (human)"]. https://www.ncbi.nlm.nih.gov/gene/1719. 
  4. ↑ "Porcine liver dihydrofolate reductase. Purification, properties, and amino acid sequence". The Journal of Biological Chemistry 254 (22): 11475–84. November 1979. doi:10.1016/S0021-9258(19)86510-9. PMID 500653. 
  5. ↑ Krahn, JM; Jackson, MR; DeRose, EF; Howell, EE; London, RE (25 December 2007). "Crystal structure of a type II dihydrofolate reductase catalytic ternary complex.". Biochemistry 46 (51): 14878–88. doi:10.1021/bi701532r. PMID 18052202. 
  6. ↑ 6.0 6.1 "Entrez Gene: DHFR dihydrofolate reductase". https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1719. 
  7. ↑ 7.0 7.1 "Structure, dynamics, and catalytic function of dihydrofolate reductase". Annual Review of Biophysics and Biomolecular Structure 33 (1): 119–40. 2004. doi:10.1146/annurev.biophys.33.110502.133613. PMID 15139807. 
  8. ↑ "Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity". Proceedings of the National Academy of Sciences of the United States of America 77 (7): 4216–20. July 1980. doi:10.1073/pnas.77.7.4216. PMID 6933469. Bibcode: 1980PNAS...77.4216U. 
  9. ↑ "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". The Journal of Biological Chemistry 284 (41): 28128–36. October 2009. doi:10.1074/jbc.M109.041483. PMID 19666465. 
  10. ↑ "Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate". Science 197 (4302): 452–5. July 1977. doi:10.1126/science.17920. PMID 17920. Bibcode: 1977Sci...197..452M. 
  11. ↑ 11.0 11.1 "Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution. II. Environment of bound NADPH and implications for catalysis". The Journal of Biological Chemistry 257 (22): 13663–72. November 1982. doi:10.1016/S0021-9258(18)33498-7. PMID 6815179. 
  12. ↑ 12.0 12.1 "Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism". Biochemistry 40 (33): 9846–59. August 2001. doi:10.1021/bi010621k. PMID 11502178. 
  13. ↑ 13.0 13.1 13.2 13.3 "How dihydrofolate reductase facilitates protonation of dihydrofolate". Journal of the American Chemical Society 125 (29): 8718–9. July 2003. doi:10.1021/ja035272r. PMID 12862454. 
  14. ↑ 14.0 14.1 "Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography". Proceedings of the National Academy of Sciences of the United States of America 111 (51): 18225–30. December 2014. doi:10.1073/pnas.1415856111. PMID 25453083. Bibcode: 2014PNAS..11118225W. 
  15. ↑ 15.0 15.1 "Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100". Proceedings of the National Academy of Sciences of the United States of America 111 (51): 18231–6. December 2014. doi:10.1073/pnas.1415940111. PMID 25453098. Bibcode: 2014PNAS..11118231L. 
  16. ↑ 16.0 16.1 "Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis". Biochemistry 50 (3): 367–75. January 2011. doi:10.1021/bi1016843. PMID 21138249. 
  17. ↑ 17.0 17.1 17.2 "Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli". Biochemistry 26 (13): 4085–92. June 1987. doi:10.1021/bi00387a052. PMID 3307916. 
  18. ↑ "Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications". Biochemistry 34 (8): 2710–23. February 1995. doi:10.1021/bi00008a039. PMID 7873554. 
  19. ↑ 19.0 19.1 19.2 "Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence". Biochemistry 36 (3): 586–603. January 1997. doi:10.1021/bi962337c. PMID 9012674. 
  20. ↑ "Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications". Biochemistry 33 (23): 7021–6. June 1994. doi:10.1021/bi00189a001. PMID 8003467. 
  21. ↑ "Role of water in the catalytic cycle of E. coli dihydrofolate reductase". Protein Science 11 (6): 1442–51. June 2002. doi:10.1110/ps.5060102. PMID 12021443. 
  22. ↑ "Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase". Biochemistry 44 (51): 16835–43. December 2005. doi:10.1021/bi051378i. PMID 16363797. 
  23. ↑ "A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site". Nature Structural Biology 2 (11): 1018–25. November 1995. doi:10.1038/nsb1195-1018. PMID 7583655. 
  24. ↑ "Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein". Biochemistry 35 (35): 11414–24. September 1996. doi:10.1021/bi960205d. PMID 8784197. 
  25. ↑ "Mechanistic studies of R67 dihydrofolate reductase. Effects of pH and an H62C mutation". The Journal of Biological Chemistry 272 (4): 2252–8. January 1997. doi:10.1074/jbc.272.4.2252. PMID 8999931. 
  26. ↑ "The tail wagging the dog: insights into catalysis in R67 dihydrofolate reductase". Biochemistry 49 (42): 9078–88. October 2010. doi:10.1021/bi1007222. PMID 20795731. 
  27. ↑ "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency". American Journal of Human Genetics 88 (2): 216–25. February 2011. doi:10.1016/j.ajhg.2011.01.004. PMID 21310276. 
  28. ↑ Nyhan, William L; Hoffmann, Georg F.; Barshop, Bruce A (30 December 2011). Atlas of Inherited Metabolic Diseases 3E. CRC Press. pp. 141–. ISBN 978-1-4441-4948-7. https://books.google.com/books?id=vCvSBQAAQBAJ&pg=PA141. 
  29. ↑ "Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi". Molecular and Cellular Biology 9 (11): 5182–8. November 1989. doi:10.1128/mcb.9.11.5182. PMID 2601715. 
  30. ↑ "Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs". Journal of Molecular Biology 295 (2): 307–23. January 2000. doi:10.1006/jmbi.1999.3328. PMID 10623528. 
  31. ↑ "The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake". Proceedings of the National Academy of Sciences of the United States of America 106 (36): 15424–9. September 2009. doi:10.1073/pnas.0902072106. PMID 19706381. 
  32. ↑ "Modified therapy with 5-fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer". Cancer 72 (1): 37–41. July 1993. doi:10.1002/1097-0142(19930701)72:1<37::AID-CNCR2820720109>3.0.CO;2-P. PMID 8508427. 
  33. ↑ 33.0 33.1 33.2 "Dihydrofolate reductase inhibitors as antibacterial agents". Biochemical Pharmacology 71 (7): 941–8. March 2006. doi:10.1016/j.bcp.2005.10.052. PMID 16359642. 
  34. ↑ "A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site". Nature Structural Biology 2 (11): 1018–25. November 1995. doi:10.1038/nsb1195-1018. PMID 7583655. 
  35. ↑ "In search of dihydrofolate reductase". Protein Science 5 (6): 1201–8. June 1996. doi:10.1002/pro.5560050626. PMID 8762155. 
  36. ↑ "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. July 2002. doi:10.1016/S0925-4439(02)00079-0. PMID 12084458. 
  37. ↑ "Evolutionary paths to antibiotic resistance under dynamically sustained drug selection". Nature Genetics 44 (1): 101–5. December 2011. doi:10.1038/ng.1034. PMID 22179135. 
  38. ↑ "Biophysical principles predict fitness landscapes of drug resistance". Proceedings of the National Academy of Sciences of the United States of America 113 (11): E1470-8. March 2016. doi:10.1073/pnas.1601441113. PMID 26929328. Bibcode: 2016PNAS..113E1470R. 
  39. ↑ "Insights into enzyme function from studies on mutants of dihydrofolate reductase". Science 239 (4844): 1105–10. March 1988. doi:10.1126/science.3125607. PMID 3125607. Bibcode: 1988Sci...239.1105B. 
  40. ↑ 40.0 40.1 "Targeted mutations of Bacillus anthracis dihydrofolate reductase condense complex structure−activity relationships". Journal of Medicinal Chemistry 53 (20): 7327–36. October 2010. doi:10.1021/jm100727t. PMID 20882962. 
  41. ↑ "An in vivo map of the yeast protein interactome". Science 320 (5882): 1465–70. June 2008. doi:10.1126/science.1153878. PMID 18467557. Bibcode: 2008Sci...320.1465T. http://www-nmr.cabm.rutgers.edu/academics/biochem694/reading/Tarassov_et_al_2008.pdf. 
  42. ↑ Ng, Say Kong (2012). "Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker". Protein Expression in Mammalian Cells. Methods in Molecular Biology. 801. pp. 161–172. doi:10.1007/978-1-61779-352-3_11. ISBN 978-1-61779-351-6. 
  43. ↑ "Protein folding in the central cavity of the GroEL-GroES chaperonin complex". Nature 379 (6564): 420–6. February 1996. doi:10.1038/379420a0. PMID 8559246. Bibcode: 1996Natur.379..420M. 
  44. ↑ "MDM2 regulates dihydrofolate reductase activity through monoubiquitination". Cancer Research 68 (9): 3232–42. May 2008. doi:10.1158/0008-5472.CAN-07-5271. PMID 18451149. 

Further reading

External links

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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