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Literature summary for 1.3.1.2 extracted from

  • Lohkamp, B.; Voevodskaya, N.; Lindqvist, Y.; Dobritzsch, D.
    Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination (2010), Biochim. Biophys. Acta, 1804, 2198-2206.
    View publication on PubMed
Search for more literature for 1.3.1.2

Application

Application Comment Organism
drug development the enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds Sus scrofa

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli strain Tuner(DE3) Sus scrofa

Protein Variants

Protein Variants Comment Organism
C126A site-directed mutagenesis, a potential [4Fe-4S]-cluster binding residue, the mutant shows slightly increased activity compared to the wild-type enzyme Sus scrofa
H673N site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme Sus scrofa
H673Q site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme Sus scrofa
Q156E site-directed mutagenesis, [4Fe-4S]-cluster binding residue, inactive mutant Sus scrofa
R235A site-directed mutagenesis, FAD binding residue, inactive mutant Sus scrofa
R235K site-directed mutagenesis, FAD binding residue, inactive mutant Sus scrofa
S670A site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme Sus scrofa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information pH dependence and kinetics of recombinant wild-type and mutant enzymes, overview Sus scrofa
0.0011
-
 Uracil pH 7.5, 30°C, recombinant mutant C126A Sus scrofa
0.0012
-
 Uracil pH 7.5, 30°C, recombinant wild-type enzyme Sus scrofa
0.0042
-
 Uracil pH 7.5, 30°C, recombinant mutant H673Q Sus scrofa
0.0057
-
 NADPH pH 7.5, 30°C, recombinant wild-type enzyme Sus scrofa
0.0061
-
 NADPH pH 7.5, 30°C, recombinant mutant C126A Sus scrofa
0.0062
-
 NADPH pH 7.5, 30°C, recombinant mutant H673Q Sus scrofa
0.007
-
 Uracil pH 7.5, 30°C, recombinant mutant H673N Sus scrofa
0.0075
-
 NADPH pH 7.5, 30°C, recombinant mutant H673N Sus scrofa
0.0188
-
 NADPH pH 7.5, 30°C, recombinant mutant S670A Sus scrofa
0.0366
-
 Uracil pH 7.5, 30°C, recombinant mutant S670A Sus scrofa

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ iron-sulfur cluster binding, overview Sus scrofa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
dihydrofluorouracil + NADP+ Sus scrofa dihydrofluorouracil is further metabolized to 2'-fluoro-beta-alanine 5-fluorouracil + NADPH + H+
-
 ?
dihydrothymine + NADP+ Sus scrofa
-
 thymine + NADPH + H+
-
 ?
dihydrouracil + NADP+ Sus scrofa
-
 uracil + NADPH + H+
-
 ?

Organism

Organism UniProt Comment Textmining
Sus scrofa Q28943
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli by affinity chromatography and gel filtration Sus scrofa

Source Tissue

Source Tissue Comment Organism Textmining
liver
-
 Sus scrofa
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
dihydrofluorouracil + NADP+ dihydrofluorouracil is further metabolized to 2'-fluoro-beta-alanine Sus scrofa 5-fluorouracil + NADPH + H+
-
 ?
dihydrofluorouracil + NADP+ pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine 671 residue. The deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. R235 is crucial for FAD binding Sus scrofa 5-fluorouracil + NADPH + H+ dihydrofluorouracil is further metabolized to 2'-fluoro-beta-alanine ?
dihydrothymine + NADP+
-
 Sus scrofa thymine + NADPH + H+
-
 ?
dihydrouracil + NADP+
-
 Sus scrofa uracil + NADPH + H+
-
 ?
additional information the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate. The active site loop comprising residues 670-683 are observed in open and closed conformational states, overview Sus scrofa ?
-
 ?

Synonyms

Synonyms Comment Organism
dihydropyrimidine dehydrogenase
-
 Sus scrofa
DPD
-
 Sus scrofa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Sus scrofa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.068
-
 Uracil pH 7.5, 30°C, recombinant mutant H673N Sus scrofa
0.117
-
 Uracil pH 7.5, 30°C, recombinant mutant H673Q Sus scrofa
0.177
-
 Uracil pH 7.5, 30°C, recombinant mutant S670A Sus scrofa
0.242
-
 Uracil pH 7.5, 30°C, recombinant wild-type enzyme Sus scrofa
0.256
-
 Uracil pH 7.5, 30°C, recombinant mutant C126A Sus scrofa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
additional information
-
 pH dependence and kinetics of recombinant wild-type and mutant enzymes, overview Sus scrofa
7.5
-
 assay at Sus scrofa

Cofactor

Cofactor Comment Organism Structure
FAD absolutely required, R235 is crucial for FAD binding Sus scrofa
NADPH dependent on Sus scrofa

General Information

General Information Comment Organism
metabolism in mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction Sus scrofa
additional information the N-terminal half of DPD is a member of a family of FAD-containing NADPH oxidoreductases, which transfer electrons to an acceptor protein or domain through [4Fe-4S] clusters of low to very low potential Sus scrofa

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5000
-
 Uracil pH 7.5, 30°C, recombinant mutant S670A Sus scrofa
9000
-
 NADPH pH 7.5, 30°C, recombinant mutant H673N Sus scrofa
9000
-
 NADPH pH 7.5, 30°C, recombinant mutant S670A Sus scrofa
10000
-
 Uracil pH 7.5, 30°C, recombinant mutant H673N Sus scrofa
19000
-
 NADPH pH 7.5, 30°C, recombinant mutant H673Q Sus scrofa
28000
-
 Uracil pH 7.5, 30°C, recombinant mutant H673Q Sus scrofa
42000
-
 NADPH pH 7.5, 30°C, recombinant mutant C126A Sus scrofa
42000
-
 NADPH pH 7.5, 30°C, recombinant wild-type enzyme Sus scrofa
208000
-
 Uracil pH 7.5, 30°C, recombinant wild-type enzyme Sus scrofa
238000
-
 Uracil pH 7.5, 30°C, recombinant mutant C126A Sus scrofa