Dihydrofolate reductase (mammalian)

 

Dihydrofolate reductase catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by stereospecific hydride transfer from a NADPH cofactor to teh C6 atom of the pterin ring with concomitant protonation at N(5). DHFR plays a central role cell maintenance of THF reserves, which are essential for purine and thimidylate synthesis and hence for cell growth and proliferation. As DHFR is the sole source of THF, the enzyme is the Achilles heel of rapidly proliferating cells and, therefore, has been a major focus in the development of anticancer and antibacterial reagents.

 

Reference Protein and Structure

Sequence
P00374 UniProt (1.5.1.3) IPR012259 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1dhf - CRYSTAL STRUCTURES OF RECOMBINANT HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 5-DEAZOFOLATE (2.3 Å) PDBe PDBsum 1dhf
Catalytic CATH Domains
3.40.430.10 CATHdb (see all for 1dhf)
Click To Show Structure

Enzyme Reaction (EC:1.5.1.3)

dihydrofolic acid
CHEBI:15633ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
(6S)-5,6,7,8-tetrahydrofolic acid
CHEBI:15635ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: 7,8-dihydrofolate reductase, DHFR, NADPH-dihydrofolate reductase, Dihydrofolate reductase:thymidylate synthase, Dihydrofolic acid reductase, Dihydrofolic reductase, Folic acid reductase, Folic reductase, Pteridine reductase:dihydrofolate reductase, Tetrahydrofolate dehydrogenase, Thymidylate synthetase-dihydrofolate reductase,

Enzyme Mechanism

Introduction

The first step of the reaction is a tautomerisation of the dihydrofolate substrate from the keto to enol form. The second step is a stereospecific hydride transfer from the NADPH cofactor to the C(5) of dihydrofolate. This step requires a general acid to donate a proton to N(5) in E. Coli, but no analogous residue exists in the human enzyme. A proton is abstracted from the enol oxygen during hydride transfer in a second tautomerisation to regenerate the keto form. The overall product is 5,6,7,8 tetrahydrofolate (THF).

Catalytic Residues Roles

UniProt PDB* (1dhf)
Leu23 Leu22A The non polar side chain disrupts the hydrogen bond between the substrate and Glu30 sufficiently to prevent the formation of an overly stable intermediate while also allowing Glu30 to hold the substrate in the correct orientation within the active site. electrostatic stabiliser
Glu31 Glu30A The residue is thought to position the dihydrofolate substrate for optimum reactivity with the hydride donor NADPH. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Cody V et al. (2005), Acta Crystallogr D Biol Crystallogr, 61, 147-155. Understanding the role of Leu22 variants in methotrexate resistance: comparison of wild-type and Leu22Arg variant mouse and human dihydrofolate reductase ternary crystal complexes with methotrexate and NADPH. DOI:10.1107/s0907444904030422. PMID:15681865.
  2. Volpato JP et al. (2009), J Biol Chem, 284, 20079-20089. Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance. DOI:10.1074/jbc.m109.018010. PMID:19478082.
  3. Cody V et al. (2009), Biochemistry, 48, 1702-1711. Correlations of Inhibitor Kinetics forPneumocystis jiroveciiand Human Dihydrofolate Reductase with Structural Data for Human Active Site Mutant Enzyme Complexes†‡. DOI:10.1021/bi801960h. PMID:19196009.
  4. Kovalevskaya NV et al. (2005), J Biomol NMR, 33, 69-72. Solution Structure of Human Dihydrofolate Reductase in its Complex with Trimethoprim and NADPH. DOI:10.1007/s10858-005-1475-z. PMID:16222560.
  5. Klon AE et al. (2002), J Mol Biol, 320, 677-693. Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution. PMID:12096917.
  6. Park H et al. (1997), J Biol Chem, 272, 2252-2258. Mechanistic Studies of R67 Dihydrofolate Reductase. EFFECTS OF pH AND AN H62C MUTATION. DOI:10.1074/jbc.272.4.2252. PMID:8999931.
  7. Lewis WS et al. (1995), J Biol Chem, 270, 5057-5064. Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers. PMID:7890613.

Step 1.

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Catalytic Residues Roles

Residue Roles
Glu30A electrostatic stabiliser
Leu22A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday