Dihydrofolate reductase
From Proteopedia
Dihydrofolate reductase (DHFR) is an enzyme which uses the co-factor NADPH as electron donor which converts it to NADP. It catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid (folate). The folate is a form of the essential vitamin B9. Antifolate inhibitors are used in cancer therapy. DHFR forms a complex with thymidylate synthase (TS). Both enzymes participate in the biosymthesis of pyrimidine. The images at the left and at the right correspond to one representative dihydrofolate reductase structure, i.e. the crystal structure of Moritella profunda dihydrofolate reductase (3ia5).
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Structure and Function
DHFR is a ubiquitous enzyme found in all organisms. The primary physiological role of DHFR is maintenance of the intracellular levels of tetrahydrofolate, a precursor of cofactors required for the biosynthesis of purines, pyrimidines, and several amino acids.
The enzyme, which is the sole source of tetrahydrofolate catalyzes the reduction of (DHF) to 5,6,7,8-tetrahydrofolate (THF) by stereospecific hydride transfer from the cofactor to the C6 atom of the pterin ring with concomitant protonation at N5. Being the sole source of THF it is an Achilles’ heel of rapidly proliferating cells, making it an attractive target of several important anticancer and antimicrobial drugs such as methotrexate, trimethoprim, and pyrimethamine.
E. coli DHFR is a small (18 kD) protein with an α/β structure consisting of a central eight-stranded β-sheet and four flanking α-helices. The active site cleft divides the protein into two structural subdomains: the (red) and the (green). The adenosine binding subdomain is the smaller of the two subdomains and provides the binding site for the adenosine moiety of the cofactor. The major subdomain consists of ~100 residues from the N and C termini and is dominated by a set of three loops on the ligand binding face that surround the active site. In terms of sequence length, these loops make up approximately 40%–50% of the major subdomain; hence it is sometimes called the “loop” subdomain. The loops are termed Met20, F-G, and G-H loops. Hinge bending motions about Lys38 and Val88 allow the adenosine binding domain to move relative to the major domain upon binding of various ligands, resulting in closure of the active site cleft.
A movie depicting the conformational changes of DHFR during one turnover of substrate has been constructed by M. R. Sawaya and J. Kraut using six isomorphous crystallographic structures. http://chem-faculty.ucsd.edu/kraut/dhfr.html
Additional Resources
For additional information, see: Cancer
3D Structures of Dihydrofolate reductase
Native DHFR
1ohk, 1ohj – hDHFR – different crystal forms - human
3ia5 – MpDHFR – Moritella profunda
2kgk, 2qk8 – BaDHFR – Bacillus anthracis
3dfr – LcDHFR – Lactobacillus casei
2w9t – SaDHFR S1 - Staphylococcus aureus
3f8y, 3f8z, 3f91, 3eig, 1dlr, 1dls – hDHFR (mutant)
2ith, 1vdr – HvDHFR - Haloferax volcanii
2rh2, 2gqv, 1vie, 1vif, 1dfr – EcDHFR – Escherichia coli
1dhi, 1dhj – EcDHFR (mutant)
1dyr – PcDHFR - Pneumocystis carinii
1zdr – BsDHFR – Bacillus stearothermophilus
1juv – DHFR – Bacteriophage T4
1ai9 – CaDHFR - Candida albicans
1cz3, 1d1g – DHFR – Thermotoga maritima
DHFR-Thymidine synthase (TS)_complex
3cl9, 2h2q – TcDHFR-TS - Trypanosoma cruzi
1qzf - Ch TcDHFR-TS – Cryptosporidium hominis
3dl5, 3dl6, 2oip – ChDHFR-TS (mutant) – Cryptosporidiium hominis
DHFR+antifolate inhibitors
3i8a, 3f0q, 3f0s, 3f0v, 3f0x, 3f0b, 3f0u, 3fq0, 3fqc, 3fqf, 3fqo, 3fqv, 3fqz – SaDHFR (mutant)+NADPH+2,4-diamino-5-(3-(2,5-dimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine
3fra – SaDHFR (mutant)+iclaprim
3frf – SaDHFR (mutant)+iclaprim+NADPH
3frb – SaDHFR (mutant)+trimethoprim (TMP)+NADP
3fre– SaDHFR (mutant)+TMP+NADPH
2w3a– hDHFR +TMP+NADPH
2w3b – hDHFR+NADPH+6-((2,5-diethoxyphenyl)aminomethyl)-2,4-diamino-methylpyrido(2,3-D)pyrimidine
2w3v– MaDHFR +TMP+NADPH – Mycobacterium avium
2w3w – MaDHFR+NADPH+6-((2,5-diethoxyphenyl)aminomethyl)-2,4-diamino-methylpyrido(2,3-D)pyrimidine
3gyf – hDHFR+Z-isomer of 2,4-diaminofuro[2,3d]pyrimidine+NADPH
3k45, 3k47 – mDHFR+E & Z-isomer+NADPH – mouse
3gi2, 3ghc, 3ghv, 3ghw – hDHFR (mutant)+2-amino-4-oxo-5,6-ethylthieno[2,3-d]pyrimidine
3fy8, 3fyw – SaDHFR + AR-101+NADPH
3fy9 – SaDHFR (mutant)+ AR-102+NADPH
3fyv – SaDHFR (mutant)+ AR-102+NADPH
3fyw – SaDHFR + AR-101+NADPH
3ia4 – MpDHFR+methotrexate (MTX)+NADPH
3hbb – TcDHFR+MTX & TMTX
3fl8 – BaDHFR+RAB1
3fl9 – BaDHFR+trimethoprim
3jw3, 3jw5 – BaDHFR (mutant)+trimethoprim+NADPH
3dat - BaDHFR+ MTX+NADPH
3e0b, 3jvx, 3jwc, 3jwf, 3jwk, 3jwm – BaDHFR (mutant)+NADP+ pyrimidine derivative
3lg4 - SaDHFR (mutant)+NADP+ pyrimidine derivative
2w9g - SaDHFR+trimethoprim+NADPH
2w9h – SaDHFR+trimethoprim
3m08 - SaDHFR+RAB1+NADP
3m09 – SaDHFR (mutant)+RAB1+NADP
2hm9 – LcDHFR+trimethoprim
1ao8 - LcDHFR+MTX – NMR
3clb – TcDHFR-TS + trimetrexate
1j3i - PfDHFR-TS+NADPH+dUMP+WR99210 – Plasmodium falciparum
1j3k - PfDHFR-TS (mutant)+NADPH+dUMP+WR99210
1j3j - PfDHFR-TS (mutant)+NADPH+dUMP+pyrimethamine
3cse, 3eej, 3eek, 3eel, 3eem - CgDHFR +NADPH+2,4-diamino-5-(3-(2,5-dimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine – Candida glabrata
3d80, 3d84 – mDHFR+NADPH+2,4-diamino-6-(2’-hydrosydibenz[b,f]azepin-5-yl)methylpteridine
1dre, 1rb3, 1rg7, 1rh3, 1ra3 - EcDHFR+NADP+MTX
1rx3 - EcDHFR+NADPH+MTX
1dra, 1drb, 2drc, 3drc, 1dds - EcDHFR+MTX
1ddr - EcDHFR+MTX+urea
1tdr - EcDHFR+MTX+Te
1rc4, 1dyj - EcDHFR+NADP+5,10-dideazatetrahydrofolate
1dyh - EcDHFR+NADP+dideazafolic acid
4dfr - EcDHFR+MTX
2c2s, 2c2t - hDHFR+ NADPH+boron containing pyrimidine
2inq – EcDHFR+MTX – neutron diffraction
1rx4 - EcDHFR+5,10-dideazatetrahydrofolate+monophosphoadenosine 5’-diphosphoribose
1rx5 - EcDHFR+5,10-dideazatetrahydrofolate
1rx6 - EcDHFR+5,10-dideazatetrahydrofolate+NADPH
1drh - EcDHFR+5,10-dideazatetrahydrofolate+folate+5-deazafolate
2fzh – PcDHFR+NADPH+ trimethoprim
1ly3, 1s3y – PcDHFR +NAP+quinazoline derivatives
1ly4 - PcDHFR +NAP+pyrimidine derivative
2fzi, 2fzj - mDHFR+NADPH+ trimethoprim
2ano, 2anq - EcDHFR+NADPH+MS-SH08-17
1yho - hDHFR+NADPH+ trimethoprim – NMR
1lud - LcDHFR+NADPH+ trimethoprim – NMR
1dis, 1diu - LcDHFR+ brodimoprim – NMR
1bzf - LcDHFR+NADPH+ TMX – NMR
2bl9, 2bla, 2blb, 2blc – PvHDFR+pyrimethamine – Plasmodium vivax
1u70 – mDHFR (mutant)+MTX+NADPH
1u71, 1u72 - hDHFR (mutant)+MTX+NADPH
1s3u, 1s3v, 1s3w – hDHFR+tetrahydroauinazoline
1mvs, 1mvt, 1pd8, 1pd9, 1pdb, 1mvs, 1mvt, 3fs6, 1hfp, 1hfq, 1hfr, 2c2s, 2c2t, 2w3b, 3gyf, 3nxo, 3nxr, 3nxt, 3nxv – hDHFR+pyrimidine derivatives
1m79, 1m78, 1m7a, 1ia1, 1ia2, 1ia3, 1ia4, 1aoe – CaDHFR+NADPH+quinazoline derivatives
1boz – hDHFR (mutant)+NADPH+quinazoline derivatives
1kms, 1kmv – hDHFR+NADPH+lipophilic antifolate
1vj3 – PcHDFR+TAB+NADPH – Plasmodium carinii
1daj – PcDHFR+NADPH+furo[2,3-d]pyrimidine
1df7 - MtDHFR +MTX+NADPH – Mycobacterium tuberculosis
1dg5 - MtDHFR + trimethoprim +NADPH
2cig – MtDHFR+isoniazid derivative
1dg7 - MtDHFR + 4-Br-WR99210 +NADPH
1dr1, 1dr2, 1dr3 - cDHFR+NADP+biopterin – chicken
1dr4, 1dr5, 1dr6, 1dr7 - cDHFR+NADP+biopterin+Hg
1klk – DHFR+NADPH+PT653 – rat
1sej - DHFR +NADPH+quinazoline derivative+deoxyuridine – Cryptosporidium hominis
3ix9 – DHFR (mutant) +MTX+NADPH – Streptococcus pneumoniae
DHFR+ligands
3frd – SaDHFR (mutant)+folate +NADP
2w3m – hDHFR +folate+NADPH
1dhf, 2dhf – hDHFR+folate+5-deazofolate
1e26 - PcDHFR-TS+NADP+folate
2jyb – HvDHFR+folate
2rk1 – EcDHFR+NADP+dihydrofolate
7dfr - EcDHFR+NAP+NADP+folate
1ra8, 1rx8 – EcDHFR+folate+monophosphoadenosine 5’-diphosphoribose
1rb2, 1rx2, 1ra2 - EcDHFR+folate+NADP
1rx7, 1rd7, 1re7 - EcDHFR+folate
1rf7 - EcDHFR+dihydrofolate
1dyi - EcDHFR+NADP+folic acid
2cd2, 3cd2, 4cd2 - PcDHFR+NADP+folate
1cd2 - PcHDFR+folate+NADPH
2zza – MpDHFR+ folate +NADP
DHFR+co-factors
2hqp – LcDHFR+NADPH – NMR
2rk2, 2p4t, 1ra9, 1rx9 – EcDHFR+NADP
1rx1, 1ra1 - EcDHFR+NADPH
6dfr - EcDHFR+NAP+NADP
1dg8 - MtDHFR +NADPH
8dfr - cDHFR +NADPH
3k74 – EcDHFR+nanobody
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