Dihydrofolate reductase

From Proteopedia

Revision as of 09:50, 11 August 2010

Template:STRUCTURE 3ia5

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. crystal structure of Moritella profunda dihydrofolate reductase (3ia5).

The sole source of tetrahydrofolate

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

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

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 3w3w – 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 2w9g - SaDHFR+trimethoprim+NADPH 2w9h – SaDHFR+trimethoprim 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 - 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 - EcDHFR+MTX 1rc4, 1dyj - EcDHFR+NADP+5,10-dideazatetrahydrofolate 1dyh - EcDHFR+NADP+dideazafolic acid 2c2s, 2c2t - hDHFR+ NADPH+boron containing pyrimidine 2inq – EcDHFR+MTX – neutron diffraction 2fzh – PcDHFR+NADPH+ trimethoprim 2fzi, 2fzj - mDHFR+NADPH+ trimethoprim 2ano, 2anq - EcDHFR+NADPH+MS-SH08-17 1yho - hDHFR+NADPH+ trimethoprim – NMR 1lud - LcDHFR+NADPH+ trimethoprim – NMR 1bzf - LcDHFR+NADPH+ TMX – NMR 2bl9 – PvHDFR+pyrimethamine – Plasmodium vivax 1u70 – mDHFR (mutant)+MTX+NADPH 1u71, 1u72 - hDHFR (mutant)+MTX+NADPH 1s3u, 1s3v, 1s3w – hDHFR+tetrahydroauinazoline 1vj3 – PcHDFR+TAB+NADPH – Plasmodium carinii 1daj – PcDHFR+NADPH+furo[2,3-d]pyrimidine 1pd8, 1pd9,1pdb – hDHFR+5-methyl-6-N-methylanilino pyridopyrimidine 1mvs, 1mvt – hDHFR+pyrido[2,3-d]pyrimidine 1m79, 1m78, 1m7a, 1ia1, 1ia2, 1ia3, 1ia4, 1aoe – CaDHFR+NADPH+quinazoline derivatives 1kms, 1kmv – hDHFR+NADPH+lipophilic antifolate 1df7 - MtDHFR +MTX+NADPH – Mycobacterium tuberculosis 1dg5 - MtDHFR + trimethoprim +NADPH 1dg7 - MtDHFR + 4-Br-WR99210 +NADPH 1hfp, 1hfq, 1hfr – hDHFR+NADPH+furopyrimidine 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

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

DHFR+co-factors

2hqp – LcDHFR+NADPH – NMR 1rx9 - EcDHFR+NADP 2rk2, 2p4t, 1ra9 – EcDHFR+NADP 1rx1, 1ra1 - EcDHFR+NADPH 6dfr - EcDHFR+NAP+NADP 1dg8 - MtDHFR +NADPH