Dihydrofolate reductase (type II)

 

R67-plasmid encoded dihydrofolate reductase (R67-DHFR) from Escherichia coli catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of NADPH. It is one of the smallest enzymes known to self-assemble into an active quaternary structure. The tetramer has an unusual pore, 25 angstroms in length that passes through the middle of the molecule and out the other side. R67-DHFR has 222 symmetry, and has a distinctive 'one site fits both' substrate and cofactor.

 

Reference Protein and Structure

Sequence
P00383 UniProt (1.5.1.3) IPR009159 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1vie - STRUCTURE OF DIHYDROFOLATE REDUCTASE (1.7 Å) PDBe PDBsum 1vie
Catalytic CATH Domains
2.30.30.60 CATHdb (see all for 1vie)
Click To Show Structure

Enzyme Reaction (EC:1.5.1.3)

NADPH(4-)
CHEBI:57783ChEBI
+
hydron
CHEBI:15378ChEBI
+
dihydrofolate(2-)
CHEBI:57451ChEBI
→
(6S)-5,6,7,8-tetrahydrofolate(2-)
CHEBI:57453ChEBI
+
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 mechanism of R67-DHFR relies on a pre-protonated form of DHF being the substrate. The substrate then accepts a hydride from NADPH to form the product, THF. The catalytic residues Tyr 69, Ile 68 and Gln 67 destabilise the ground state. Lys 32 is involved in stabilising the transition state.

Catalytic Residues Roles

UniProt PDB* (1vie)
Lys32 Lys32(16)A The positively charged sidechain of Lys 32 helps to stabilise the negatively charged intermediate. electrostatic stabiliser
Gln67 Gln67(51)A Gln 67 acts to destabilise the ground state of the substrate. electrostatic stabiliser
Ile68 Ile68(52)A Ile 68 acts to destabilise the ground state. electrostatic stabiliser
Tyr69 Tyr69(53)A Tyr 69 acts to destabilise the ground state. 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

hydride transfer, aromatic unimolecular elimination by the conjugate base, overall product formed, overall reactant used, bimolecular nucleophilic addition, cofactor used

References

  1. Strader MB et al. (2001), Biochemistry, 40, 11344-11352. Role of S65, Q67, I68, and Y69 Residues in Homotetrameric R67 Dihydrofolate Reductase†. DOI:10.1021/bi0110544. PMID:11560482.
  2. Timson MJ et al. (2013), Biochemistry, 52, 2118-2127. Further studies on the role of water in R67 dihydrofolate reductase. DOI:10.1021/bi301544k. PMID:23458706.
  3. Krahn JM et al. (2007), Biochemistry, 46, 14878-14888. Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. DOI:10.1021/bi701532r. PMID:18052202.
  4. Howell EE (2005), Chembiochem, 6, 590-600. Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis. DOI:10.1002/cbic.200400237. PMID:15812782.

Step 1. A hydride is transferred from NADPH to the protonated form of DHF, forming the product and NADP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ile68(52)A electrostatic stabiliser
Lys32(16)A electrostatic stabiliser
Tyr69(53)A electrostatic stabiliser
Gln67(51)A electrostatic stabiliser

Chemical Components

hydride transfer, ingold: aromatic unimolecular elimination by the conjugate base, overall product formed, overall reactant used, ingold: bimolecular nucleophilic addition, cofactor used
Download: Image, Marvin File

Step 1. A hydride is transferred from NADPH to the protonated form of DHF, forming the product and NADP.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Amelia Brasnett