Involved in pyrimidine base degradation. Catalyzes the reduction of uracil and thymine. Also involved the degradation of the chemotherapeutic drug 5-fluorouracil.
Interacting selectively and non-covalently with a 4 iron, 4 sulfur (4Fe-4S) cluster; this cluster consists of four iron atoms, with the inorganic sulfur atoms found between the irons and acting as bridging ligands.
OBJECTIVE: Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism. METHODS: This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity. RESULTS: Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity. CONCLUSION: Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.
J. Biol. Chem. 267, 17102-17109 (1992)[PubMed:1512248]
Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
Evidence
4:
Inferred from Mutant PhenotypeUniProtKB
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.
J. Chromatogr. B Biomed. Sci. Appl. 729, 307-314 (1999)[PubMed:10410956]
A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2-(14)C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.
Interacting selectively and non-covalently with FAD, flavin-adenine dinucleotide, the coenzyme or the prosthetic group of various flavoprotein oxidoreductase enzymes, in either the oxidized form, FAD, or the reduced form, FADH2.
Interacting selectively and non-covalently with nicotinamide-adenine dinucleotide phosphate, a coenzyme involved in many redox and biosynthetic reactions; binding may be to either the oxidized form, NADP+, or the reduced form, NADPH.
J. Biol. Chem. 267, 17102-17109 (1992)[PubMed:1512248]
Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
The chemical reactions and pathways resulting in the formation of beta-alanine (3-aminopropanoic acid), an achiral amino acid and an isomer of alanine. It occurs free (e.g. in brain) and in combination (e.g. in pantothenate) but it is not a constituent of proteins.
The chemical reactions and pathways resulting in the breakdown of purine nucleobases, one of the two classes of nitrogen-containing ring compounds found in DNA and RNA, which include adenine and guanine.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.
The chemical reactions and pathways resulting in the breakdown of thymidine, deoxyribosylthymine thymine 2-deoxyriboside, a deoxynucleoside very widely distributed but occurring almost entirely as phosphoric esters in deoxynucleotides and deoxyribonucleic acid, DNA.
J. Biol. Chem. 267, 17102-17109 (1992)[PubMed:1512248]
Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
The chemical reactions and pathways resulting in the breakdown of thymine, 5-methyluracil, one of the two major pyrimidine bases present (as thymidine) in DNA but not found in RNA other than (as ribothymidine) in transfer RNA, where it is a minor base.
J. Chromatogr. B Biomed. Sci. Appl. 729, 307-314 (1999)[PubMed:10410956]
A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2-(14)C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.
The chemical reactions and pathways resulting in the breakdown of uracil, 2,4-dioxopyrimidine, one of the pyrimidine bases occurring in RNA, but not in DNA.
OBJECTIVE: Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism. METHODS: This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity. RESULTS: Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity. CONCLUSION: Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.
J. Biol. Chem. 267, 17102-17109 (1992)[PubMed:1512248]
Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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