User:Gisele A. Andree/Sandbox 1

From Proteopedia

Structure of Eukaryotic Dihydropyrimidine Dehydrogenase

Dihydropyrimidine Dehydrogenase (DPD) is an important enzyme involved in the degradation of pyrimidines in the body. It is a 220 kDa protein that binds co-factors; FAD, FMN, [4Fe-4S] clusters, and substrates; NADPH and pyrimidines.

Function

Dihydropyrimidine Dehydrogenase (DPD) is the first enzyme in pyrimidine degradation pathway. It catalyzes the reduction of 5,6-double bond to obtain dihydropyrimidine.

Reduction of Uracil to form 5,6-dihydro uracil. This reaction is catalyzed by eukaryotic DPD. Other pyrimidines can take the place of uracil in this reaction and will be metabolized in the same way.

Disease

5-fluorouracil (5-FU) is a drug used to treat a variety of cancers as it has wide anti-tumor activity & works well alongside other chemotherapy drugs. In the human liver 80-85% of 5-FU is catabolized into inactive, and potentially toxic, metabolites by DPD.

Only 1-3% of the original dose proceeds through anabolic pathways to create active cytotoxic complexes. The active complexes inhibit DNA synthesis and the processing and function of RNA processing thus producing a deleterious effect on both healthy and cancerous cells.

DPD decreases effectivity of drug thus requires a very high dosages, leading to major side effects. Luckily, inhibitors are in development and some are in clinical trials.

Structural highlights

, with each monomer consisting of five domains.

Domain 1 Consists of residues 27-173. It binds 2 Fe-S clusters and is made of exclusively alpha-helices. One of the [4Fe-4S] clusters is odd as it has a different coordination that is not observed in other Fe-S proteins. Three Fe atoms interact with cysteines 91, 130 and 136, but the fourth Fe atom interacts with the Oε1 glutamine 156. Since the oxygen has a smaller atomic radius than sulfur, so the Fe-O distance is much shorter (~2.0 Å) than the average Fe-S interactions seen in DPD (2.3 Å).

Domain 2 and 3 Domain 2 consists of residues 173-286 and 442-524. It binds FAD and NADPH. It contains a central parallel beta-sheet surrounded by alpha helices, forming Rossman-type nucleotide binding motifs that bind FAD and NADPH.

Domain 3 consists of residues 287-441 and also binds FAD and NADPH. Just like domain 2, contains a central parallel beta-sheet surrounded by alpha helices, forming Rossman-type nucleotide binding motifs that bind FAD and NADPH. But, D3 also contains an additional 3 stranded antiparallel beta-sheet. With the aforementioned exception, D2 and D3 are so similar that they are thought to have originated from gene duplication.

In the following figure, domain 2 is shown in blue, domain 3 is in green, FAD is colored in orange and NADPH is in pink.

Domain 4 Domain 4 has a typical α/β 8 barrel fold. It binds FMN towards the C-terminal end of the barrel forming strands. Most of the residues that have their side chains interacting with FMN are conserved between homologous structures. Two lysine residues (K574 and K709) bind to the isoalloxazine ring. Positively charged lysine 574 interacts with the redox active N5 atom of FMN ring.

Domain 5 Domain 5 consists of residues 1-26 and 848-1020. It also binds two [4Fe-4S] clusters. Between these clusters there are two α-helices and a four-stranded antiparallel β-sheet. The [4Fe-4S] clusters here in domian 5, which act as electron transfer centers, are not close to the next active redox site (the FMN binding site in domain 4). There is a gap of ~23 Å between N-terminal domain 5 Fe-S clusters and the FMN in the domian 4 binding site, which is too long for electrons to be able to cross. However, in dimer confirmation, the redox partners are close enough for electrons to transfer, thus, the enzyme must be in dimer confirmation to be active.

Domain 5 is shown in cyan and domain 4 is shown in pink. The distance between the [4Fe-4S] clusters in domain 5 and the bound FMN (yellow) in domain 4 is indicated.

Electron Transport Chain

The enzyme has a two-site ping-pong mechanism with NADPH reducing FAD and reduced FMN responsible for reducing the pyrimidine. Transfer of electrons from NADPH to the pyrimidine is downhill with FMN rapidly reduced by FADH2 via the Fe-S conduit. Two single electrons are transferred via the Fe-S pathways. The reduction of the pyrimidine at the second active proceeds using general acid catalysis with protonation at N5 of FMN carried out by lysine-574, as FMN is reduced, and protonation at C5 of the pyrimidine by Cysteine-671, as it is reduced.

As earlier state, the dimer configuration is vitally important to the enzyme's function as electrons cannot travel through the Fe-S pathway without crossing over the dimer interface. In the isolated monomers, the gap between the N-terminal Fe-S clusters and FMN about 23 Å apart, too far for electrons to cross. As a result, all 12 redox co-factors of the DPD dimer are organized into two electron transfer chains passing the dimer interface twice. In this structural configuration, all redox partners are separated by ~7.5-10 Å, distances that are commonly observed in other multicenter electron transfer chains. Thus, DPD can only be active if it is in a dimer configuration and all of the Fe-S clusters participate in the electron transfer.

References

<Dobritzsch, D., Schneider, G., Schnackerz, K. D., & Lindqvist, Y. (2001). Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti‐cancer drug 5‐fluorouracil. The EMBO journal, 20(4), 650-660./> <Schnackerz, K. D., Dobritzsch, D., Lindqvist, Y., & Cook, P. F. (2004). Dihydropyrimidine dehydrogenase: a flavoprotein with four iron–sulfur clusters. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1701(1-2), 61-74./>