Dihydrofolate reductase (bacterial)

 

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 the 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.

Much interest has been generated in this enzyme due to its potential as a target for antibacterial and anticancer drugs. It is ubiquitous throughout evolution, but always most important in cells which are dividing rapidly, hence its value as a drug target against infections and cancer.

 

Reference Protein and Structure

Sequence
P00381 UniProt (1.5.1.3) IPR012259 (Sequence Homologues) (PDB Homologues)
Biological species
Lactobacillus casei (Bacteria) Uniprot
PDB
3dfr - CRYSTAL STRUCTURES OF ESCHERICHIA COLI AND LACTOBACILLUS CASEI DIHYDROFOLATE REDUCTASE REFINED AT 1.7 ANGSTROMS RESOLUTION. I. GENERAL FEATURES AND BINDING OF METHOTREXATE (1.7 Å) PDBe PDBsum 3dfr
Catalytic CATH Domains
3.40.430.10 CATHdb (see all for 3dfr)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:1.5.1.3)

NADPH
CHEBI:16474ChEBI
+
dihydrofolic acid
CHEBI:15633ChEBI
+
hydron
CHEBI:15378ChEBI
→
5,6,7,8-tetrahydrofolic acid
CHEBI:20506ChEBI
+
NADP(+)
CHEBI:18009ChEBI
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

This mechanism proceeds in a single step in which the hydride ion is added to C6 with concomitant protonation of the N5 from a conserved water molecule. The active site residues are responsible for maintaining the steric placement of the substrates and Asp26, shown to be critical to the mechanism, is part of a hydrogen bonding network that modifies the pKa of the N5 atom from 2.4 to 6.5 when bound in a ternary complex. Further, and somewhat unusually, catalysis in this enzyme is dependent on a number of non-polar residues.

Catalytic Residues Roles

UniProt PDB* (3dfr)
Trp22 Trp21A Helps bind and activate the water molecule that (along with Asp26) helps modify the pKa of the N5. activator, hydrogen bond donor
Asp27 Asp26A the ionised Asp26 contributes to a negative electrostatic environment that shifts the solution pKa of the substrate N5 atom from 2.4 to 6.5 when bound in a ternary complex. In addition, Asp26 interacts with structurally relevant waters at the active site that are important for substrate binding. Site-directed mutagenesis of Asp26 to asparagine has demonstrated its importance for catalysis, although in eukaryotic enzymes it is replaced by a Glu residue. modifies pKa, hydrogen bond acceptor
Thr117 Thr116A Stabilises the negative charge on Asp26. hydrogen bond donor, electrostatic stabiliser
Leu95 (main-C), Leu5 (main-C) Leu94A (main-C), Leu4A (main-C) Two mainchain carbonyls, of Leu4 and Leu94, constrain the pteridine ring in the right plane to receive protons; the sidechains of these residues are not conserved, although they are always hydrophobic. steric role
Leu20 Leu19A Leu19 (replaced by Met in E. coli) may create a hydrophobic environment around N5 of folate in order to push the positive charge on the ring on to C6, where it can receive hydride. enhance reactivity
Phe31 Phe30A Phe30 sterically forces the pteridine ring close to the nicotinamide. steric locator
Leu28, Leu55 Leu27A, Leu54A Leu27 and Leu54 are both suggested to constrain the folate ring system. steric locator
*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

proton transfer, overall reactant used, intermediate formation, hydride transfer, bimolecular nucleophilic addition

References

  1. Liu CT et al. (2014), Proc Natl Acad Sci U S A, 111, 18231-18236. Escherichia colidihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100. DOI:10.1073/pnas.1415940111. PMID:25453098.
  2. Wan Q et al. (2014), Proc Natl Acad Sci U S A, 111, 18225-18230. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. DOI:10.1073/pnas.1415856111. PMID:25453083.
  3. Czekster CM et al. (2011), Biochemistry, 50, 367-375. Kinetic and Chemical Mechanism of the Dihydrofolate Reductase fromMycobacterium tuberculosis. DOI:10.1021/bi1016843. PMID:21138249.
  4. Shrimpton P et al. (2002), Protein Sci, 11, 1442-1451. Role of water in the catalytic cycle ofE. colidihydrofolate reductase. DOI:10.1110/ps.5060102. PMID:12021443.
  5. Cummins PL et al. (2001), J Am Chem Soc, 123, 3418-3428. Energetically Most Likely Substrate and Active-Site Protonation Sites and Pathways in the Catalytic Mechanism of Dihydrofolate Reductase. DOI:10.1021/ja0038474. PMID:11472112.
  6. Casarotto MG et al. (1999), Biochemistry, 38, 8038-8044. Direct Measurement of the pKaof Aspartic Acid 26 inLactobacillus caseiDihydrofolate Reductase:  Implications for the Catalytic Mechanism. DOI:10.1021/bi990301p. PMID:10387048.
  7. Brown KA et al. (1992), Faraday Discuss, 93, 217-224. Exploring the molecular mechanism of dihydrofolate reductase. DOI:10.1039/fd9929300217. PMID:1290933.
  8. Bystroff C et al. (1990), Biochemistry, 29, 3263-3277. Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state. DOI:10.2210/pdb6dfr/pdb. PMID:2185835.
  9. Bolin JT et al. (1982), J Biol Chem, 257, 13650-13662. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. PMID:6815178.

Step 1. The dihydrofolate substrate undergoes a tautomerisation reaction, resulting in the deprotonation of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr116A hydrogen bond donor, electrostatic stabiliser
Asp26A hydrogen bond acceptor
Trp21A hydrogen bond donor, activator
Leu19A enhance reactivity
Phe30A steric locator
Leu27A steric locator
Leu54A steric locator
Leu4A (main-C) steric role
Leu94A (main-C) steric role
Asp26A modifies pKa

Chemical Components

proton transfer, overall reactant used, intermediate formation, hydride transfer, ingold: bimolecular nucleophilic addition
Download: Image, Marvin File

Step 1. The dihydrofolate substrate undergoes a tautomerisation reaction, resulting in the deprotonation of water.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Alex Gutteridge, Craig Porter