Dihydropyrimidine dehydrogenase (NADP+)

 

Mammalian dihydropyrimidine dehydrogenase catalyses the reduction of uracil or thymine to dihydrouracil or dihydrothymine respectively. This reaction represents an important step in the pathway of pyrimidine degradation in cells, but is particularly important to medicine because the anticancer drug 5-flourouracil, though shown to be effective, is also a substrate for this enzyme, thus its effectiveness is reduced. The development of specific inhibitors is therefore paramount in order to reduce the cost and side effects of treatment with this drug.

 

Reference Protein and Structure

Sequence
Q28943 UniProt (1.3.1.2) IPR005720 (Sequence Homologues) (PDB Homologues)
Biological species
Sus scrofa (pig) Uniprot
PDB
1h7x - Dihydropyrimidine dehydrogenase (DPD) from pig, ternary complex of a mutant enzyme (C671A), NADPH and 5-fluorouracil (2.01 Å) PDBe PDBsum 1h7x
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1h7x)
Cofactors
Tetra-mu3-sulfido-tetrairon (4), Fadh2(2-) (1), Fmnh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.3.1.2)

uracil
CHEBI:17568ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
+
hydron
CHEBI:15378ChEBI
→
5,6-dihydrouracil
CHEBI:15901ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: 4,5-dihydrothymine: oxidoreductase, DHPDH, DHU dehydrogenase, DPD, Dehydrogenase, dihydrouracil (nicotinamide adenine dinucleotide phosphate), Dihydrothymine dehydrogenase, Dihydrouracil dehydrogenase (NADP), Hydropyrimidine dehydrogenase, Dihydrouracil dehydrogenase (NADP+),

Enzyme Mechanism

Introduction

The enzyme uses electrons from NADPH to reduce the uracil substrate. However, the electrons are not passed directly to uracil but instead are transferred from NADPH to FAD and then from FAD to FMN. Finally the FMN acts as a nucleophile to transfer a hydride ion to the double bond of the uracil. This creates a transient carbanion at the second carbon of the double bond, allowing protonation by Cys 671 to occur, completing the reaction.

Catalytic Residues Roles

UniProt PDB* (1h7x)
Lys709, Lys574 Lys709C, Lys574C Both residues stabilise the reduced FMN. It is not known whether there is formal proton transfer or just simple ionic interactions. electrostatic stabiliser
Cys671 Ala671C Ats as general acid base to protonate the transient carbanion formed after hydride transfer to the uracil. proton acceptor, proton donor
*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, cofactor used, michael addition, overall reactant used, proton transfer, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Dobritzsch D et al. (2001), EMBO J, 20, 650-660. Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil. DOI:10.1093/emboj/20.4.650. PMID:11179210.
  2. Schnackerz KD et al. (2004), Biochim Biophys Acta, 1701, 61-74. Dihydropyrimidine dehydrogenase: a flavoprotein with four iron-sulfur clusters. DOI:10.1016/j.bbapap.2004.06.009. PMID:15450176.
  3. Rosenbaum K et al. (1998), Biochemistry, 37, 17598-17609. Porcine Recombinant Dihydropyrimidine Dehydrogenase:  Comparison of the Spectroscopic and Catalytic Properties of the Wild-Type and C671A Mutant Enzymes†. DOI:10.1021/bi9815997. PMID:9860876.

Step 1. Binding of NADPH results in a conformational change which facilitates the transfer of a hydride from its pro-S position to the re-face of FAD at N5. Following this step two single electron transfers then occur via the FeS clusters to FMN.

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, cofactor used

Step 2. The reduction of uracil occurs in two steps with hydride transfer from reduced FMN to C6 first. The reduced FMN is stabilised by Lys709 and Lys574 but it is not know whether a formal proton transfer occurs or just electrostatic interactions used. The intermediate enolate formed is stabilised by delocalisation of electrons.

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Catalytic Residues Roles

Residue Roles
Lys574C electrostatic stabiliser
Lys709C electrostatic stabiliser

Chemical Components

michael addition, hydride transfer, overall reactant used

Step 3. Cys671 acts as a general acid to protonate C5, forming the product.

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Catalytic Residues Roles

Residue Roles
Lys574C electrostatic stabiliser
Lys709C electrostatic stabiliser
Ala671C proton donor

Chemical Components

proton transfer, overall product formed

Step 4. In an inferred reaction step Cys671 is reprotonated to regenerate the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala671C proton acceptor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Binding of NADPH results in a conformational change which facilitates the transfer of a hydride from its pro-S position to the re-face of FAD at N5. Following this step two single electron transfers then occur via the FeS clusters to FMN.

Step 2. The reduction of uracil occurs in two steps with hydride transfer from reduced FMN to C6 first. The reduced FMN is stabilised by Lys709 and Lys574 but it is not know whether a formal proton transfer occurs or just electrostatic interactions used. The intermediate enolate formed is stabilised by delocalisation of electrons.

Step 3. Cys671 acts as a general acid to protonate C5, forming the product.

Step 4. In an inferred reaction step Cys671 is reprotonated to regenerate the native state of the enzyme.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Amelia Brasnett