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A. H. Van Gennip - One of the best experts on this subject based on the ideXlab platform.
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Dihydropyrimidinase deficiency and severe 5 fluorouracil toxicity
Clinical Cancer Research, 2003Co-Authors: Andre B P Van Kuilenburg, Rutger Meinsma, Koichi Matsuda, Nanaya Tamaki, Bernard A Zonnenberg, Lida Zoetekouw, Frank Baas, A. H. Van GennipAbstract:Dihydropyrimidinase (DHP) is the second enzyme in the catabolism of 5-fluorouracil (5FU), and it has been suggested that patients with a deficiency of this enzyme are at risk from developing severe 5FU-associated toxicity. In this study, we demonstrated for the first time that in one patient the severe toxicity, after a treatment with 5FU, was attributable to a partial deficiency of DHP. Analysis of the DHP gene showed that the patient was heterozygous for the missense mutation 833G>A (G278D) in exon 5. Heterologous expression of the mutant enzyme in Escherichia coli showed that the G278D mutation leads to a mutant DHP enzyme without residual activity. An analysis for the presence of this mutation in 96 unrelated Dutch Caucasians indicates that the allele frequency in the normal population is <0.5%. Our results show that a partial DHP deficiency is a novel pharmacogenetic disorder associated with severe 5FU toxicity.
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Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and Dihydropyrimidinase deficiency
Journal of Chromatography B, 2003Co-Authors: Tomiko Kuhara, Yoshiro Wada, A. H. Van Gennip, Satoshi Sumi, Chie Ohdoi, Morimasa Ohse, Tetsuya Ito, Isamu MatsumotoAbstract:A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly Dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography-mass spectrometry. A proposal to use the number (n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because "n"-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4-12 for DHT and 7.2-11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders. (C) 2003 Elsevier Science B.V. All rights reserved
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detection of β ureidopropionase deficiency with hplc electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level
Journal of Inherited Metabolic Disease, 2001Co-Authors: Ron A. Wevers, A B P Van Kuilenburg, G H Van Lenthe, Birgit Assmann, Gudrun Gohlichratmann, Georg F Hoffmann, Christa Brautigam, A. H. Van GennipAbstract:The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to β-alanine and β-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and Dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-β-amino aciduria, due to a deficiency of β-ureidopropionase, was easily diagnosed at the metabolite level using HPLC–tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of β-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of β-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and Dihydropyrimidinase was present. Thus, HPLC–tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of β-ureidopropionase.
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radiochemical assay for determination of Dihydropyrimidinase activity using reversed phase high performance liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 1999Co-Authors: A B P Van Kuilenburg, H Van Lenthe, A. H. Van GennipAbstract:Abstract 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-β-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 [2-14C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a Dihydropyrimidinase deficiency.
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clinical and biochemical aspects of Dihydropyrimidinase deficiency
Advances in Experimental Medicine and Biology, 1998Co-Authors: A. H. Van Gennip, R. A. De Abreu, P Vreken, A B P Van KuilenburgAbstract:Dihydropyrimidinase (DHP, EC 3.5.2.2) catalyzes the second step in the degradation of uracil and thymine. The first step is catalyzed by dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), the third step by β-ureidopropionase (UP, EC 3.5.1.6) and the fourth step is catalyzed by three transaminases (R)-(−)-β-aminoisobutyrate pyruvate aminotransferase (BAIBPAT, EC 2.6.1.40), β-alanine-pyruvate aminotransferase (BAPAT, EC 2.6.1.18) and β-alanine-α-ketoglutarate aminotransferase (BAKAT, EC 2.6.1.19). The first three steps of the catabolism of uracil and thymine are controlled by enzymes shared by both pathways and result in the production of the neurotransmitter acid β-alanine from uracil and the nonfunctional (R)-β-aminoisobutyrate from thymine. The thymine analogue 5-fluorouracil is degraded by the same pathway to fluoro-β-alanine. In contrast to DPD deficiency of which 50 cases have been reported1,2 only six cases have been described with DHP deficiency and none with UP deficiency,3–9 although secondary UP deficiency has been reported in patients with propionic acidemia.10 The reason for this difference may be a lesser frequency of DHP compared to DPD deficient individuals, but another possibility may be that patients with DHP deficiency are overlooked. Therefore, we will focus on the clinical presentation and biochemical detection of patients with DHP deficiency.
Cheng-yang Huang - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of Dihydropyrimidinase inhibited by plumbagin isolated from nepenthes miranda extract
Biochimie, 2020Co-Authors: Yenhua Huang, Yi Lien, Junghung Chen, Enshyh Lin, Cheng-yang HuangAbstract:Abstract Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the Dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited Dihydropyrimidinase with IC50 value of 58 ± 3 μM. Double reciprocal results of Lineweaver–Burk plot indicated that this compound is a competitive inhibitor of Dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with Dihydropyrimidinase with the Kd value of 37.7 ± 1.4 μM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in Dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of Dihydropyrimidinase caused low plumbagin binding affinity. In addition to their Dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in Dihydropyrimidinase during catalysis.
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crystal structure of Dihydropyrimidinase in complex with anticancer drug 5 fluorouracil
Biochemical and Biophysical Research Communications, 2019Co-Authors: Yenhua Huang, Zhijun Ning, Cheng-yang HuangAbstract:Abstract Dihydropyrimidinase (DHPase) catalyzes the reversible cyclization of dihydrouracil to N-carbamoyl-β-alanine in the second step of the pyrimidine degradation pathway. Whether 5-fluorouracil (5-FU), the best-known fluoropyrimidine that is used to target the enzyme thymidylate synthase for anticancer therapy, can bind to DHPase remains unknown. In this study, we found that 5-FU can form a stable complex with Pseudomonas aeruginosa DHPase (PaDHPase). The crystal structure of PaDHPase complexed with 5-FU was determined at 1.76 A resolution (PDB entry 6KLK). Various interactions between 5-FU and PaDHPase were examined. Six residues, namely, His61, Tyr155, Asp316, Cys318, Ser289 and Asn337, of PaDHPase were involved in 5-FU binding. Except for Cys318, these residues are also known as the substrate-binding sites of DHPase. 5-FU interacts with the main chains of residues Ser289 (3.0 A) and Asn337 (3.2 A) and the side chains of residues Tyr155 (2.8 A) and Cys318 (2.9 A). Mutation at either Tyr155 or Cys318 of PaDHPase caused a low 5-FU binding activity of PaDHPase. This structure and the binding mode provided molecular insights into how the dimetal center in DHPase undergoes a conformational change during 5-FU binding. Further research can directly focus on revisiting the role of DHPase in anticancer therapy.
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structural basis for ph dependent oligomerization of Dihydropyrimidinase from pseudomonas aeruginosa pao1
Bioinorganic Chemistry and Applications, 2018Co-Authors: Jenhao Cheng, Yenhua Huang, Chienchih Huang, Cheng-yang HuangAbstract:Dihydropyrimidinase, a dimetalloenzyme containing a carboxylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. Unlike all known Dihydropyrimidinases, which are tetrameric, pseudomonal Dihydropyrimidinase forms a dimer at neutral pH. In this paper, we report the crystal structure of P. aeruginosa Dihydropyrimidinase at pH 5.9 (PDB entry 5YKD). The crystals of P. aeruginosa Dihydropyrimidinase belonged to space group C2221 with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 A. The structure of P. aeruginosa Dihydropyrimidinase was solved at 2.17 A resolution. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa Dihydropyrimidinase monomers. Gel filtration chromatographic analysis of purified P. aeruginosa Dihydropyrimidinase revealed a mixture of dimers and tetramers at pH 5.9. Thus, P. aeruginosa Dihydropyrimidinase can form a stable tetramer both in the crystalline state and in the solution. Based on sequence analysis and structural comparison of the dimer-dimer interface between P. aeruginosa Dihydropyrimidinase and Thermus sp. Dihydropyrimidinase, different oligomerization mechanisms are proposed.
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crystal structure of Dihydropyrimidinase from pseudomonas aeruginosa pao1 insights into the molecular basis of formation of a dimer
Biochemical and Biophysical Research Communications, 2016Co-Authors: Chingting Tzeng, Yenhua Huang, Cheng-yang HuangAbstract:Abstract Dihydropyrimidinase, a tetrameric metalloenzyme, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. In this paper, we report the crystal structure of Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 at 2.1 A resolution. The structure of P. aeruginosa Dihydropyrimidinase reveals a classic (β/α)8-barrel structure core embedding the catalytic dimetal center and a β-sandwich domain, which is commonly found in the architecture of Dihydropyrimidinases. In contrast to all Dihydropyrimidinases, P. aeruginosa Dihydropyrimidinase forms a dimer, rather than a tetramer, both in the crystalline state and in the solution. Basing on sequence analysis and structural comparison of the C-terminal region and the dimer–dimer interface between P. aeruginosa Dihydropyrimidinase and Thermus sp. Dihydropyrimidinase, we propose a working model to explain why this enzyme cannot be a tetramer.
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Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases.
PloS one, 2015Co-Authors: Cheng-yang HuangAbstract:Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit Dihydropyrimidinase remains unclear. This study investigated the inhibition of Dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit Dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of Dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on Dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited Dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with Dihydropyrimidinase with the Kd value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of Dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited Dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases.
A B P Van Kuilenburg - One of the best experts on this subject based on the ideXlab platform.
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beta alanine and beta aminoisobutyric acid levels in two siblings with Dihydropyrimidinase deficiency
Nucleosides Nucleotides & Nucleic Acids, 2008Co-Authors: A B P Van Kuilenburg, A E M Stroomer, Annet M Bosch, M DuranAbstract:Dihydropyrimidinase (DHP) deficiency is an inborn error of the pyrimidine degradation pathway, affecting the hydrolytic ring opening of the dihydropyrimidines. In two siblings with a complete DHP deficiency and a variable clinical presentation, a normal concentration of beta-alanine and strongly decreased levels of beta-aminoisobutyric acid were observed in plasma, urine and CSF. No major differences were observed for the concentrations of the beta-amino acids in plasma and urine between the symptomatic and asymptomatic sibling. Thus, the relevance of the shortage of beta-aminoisobutyric acid for the onset of a clinical phenotype in patients with DHP deficiency remains to be established.
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detection of β ureidopropionase deficiency with hplc electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level
Journal of Inherited Metabolic Disease, 2001Co-Authors: Ron A. Wevers, A B P Van Kuilenburg, G H Van Lenthe, Birgit Assmann, Gudrun Gohlichratmann, Georg F Hoffmann, Christa Brautigam, A. H. Van GennipAbstract:The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to β-alanine and β-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and Dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-β-amino aciduria, due to a deficiency of β-ureidopropionase, was easily diagnosed at the metabolite level using HPLC–tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of β-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of β-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and Dihydropyrimidinase was present. Thus, HPLC–tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of β-ureidopropionase.
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radiochemical assay for determination of Dihydropyrimidinase activity using reversed phase high performance liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 1999Co-Authors: A B P Van Kuilenburg, H Van Lenthe, A. H. Van GennipAbstract:Abstract 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-β-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 [2-14C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a Dihydropyrimidinase deficiency.
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clinical and biochemical aspects of Dihydropyrimidinase deficiency
Advances in Experimental Medicine and Biology, 1998Co-Authors: A. H. Van Gennip, R. A. De Abreu, P Vreken, A B P Van KuilenburgAbstract:Dihydropyrimidinase (DHP, EC 3.5.2.2) catalyzes the second step in the degradation of uracil and thymine. The first step is catalyzed by dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), the third step by β-ureidopropionase (UP, EC 3.5.1.6) and the fourth step is catalyzed by three transaminases (R)-(−)-β-aminoisobutyrate pyruvate aminotransferase (BAIBPAT, EC 2.6.1.40), β-alanine-pyruvate aminotransferase (BAPAT, EC 2.6.1.18) and β-alanine-α-ketoglutarate aminotransferase (BAKAT, EC 2.6.1.19). The first three steps of the catabolism of uracil and thymine are controlled by enzymes shared by both pathways and result in the production of the neurotransmitter acid β-alanine from uracil and the nonfunctional (R)-β-aminoisobutyrate from thymine. The thymine analogue 5-fluorouracil is degraded by the same pathway to fluoro-β-alanine. In contrast to DPD deficiency of which 50 cases have been reported1,2 only six cases have been described with DHP deficiency and none with UP deficiency,3–9 although secondary UP deficiency has been reported in patients with propionic acidemia.10 The reason for this difference may be a lesser frequency of DHP compared to DPD deficient individuals, but another possibility may be that patients with DHP deficiency are overlooked. Therefore, we will focus on the clinical presentation and biochemical detection of patients with DHP deficiency.
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Dihydropyrimidinase deficiency confirmation of the enzyme defect in dihydropyrimidinuria
Journal of Inherited Metabolic Disease, 1997Co-Authors: A. H. Van Gennip, R. A. De Abreu, Jan J. Rotteveel, P Vreken, G H Van Lenthe, J A J M Bakkeren, A B P Van KuilenburgAbstract:Dihydropyrimidinase (DHP, EC 3.5.2.2) is the second enzyme in the degradation pathway of uracil and thymine. It catalyses the degradation of both dihydrouracil and dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. So far, four cases of dihydropyrimidinuria (McKusick 222748) have been reported (Duran et al 1991; Henderson et al 1993; Bakkeren et al, personal communication, 1994; Ohba et al 1994). The patients show a variable clinical phenotype comprising seizures or epileptic attacks (3 out of 4 patients) mental retardation (2 patients), growth retardation (1 patient) and dysmorphic features (1 patient). Since these patients excrete large amounts of dihydrouracil and dihydrothymine and moderate amounts of uracil and thymine in their urine, they can easily be detected (Van Gennip et al 1993). On the basis of the characteristic urinary metabolite profile it is assumed that the disease is caused by a deficiency of DHP. The direct measurement of the activity of DHP in patients has been hampered by the fact that the enzyme is almost exclusively expressed in liver tissue. Here, we provide for the first time direct evidence at the enzyme level for a deficient activity of DHP in liver in a patient with dihydropyrimidinuria.
G H Van Lenthe - One of the best experts on this subject based on the ideXlab platform.
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detection of β ureidopropionase deficiency with hplc electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level
Journal of Inherited Metabolic Disease, 2001Co-Authors: Ron A. Wevers, A B P Van Kuilenburg, G H Van Lenthe, Birgit Assmann, Gudrun Gohlichratmann, Georg F Hoffmann, Christa Brautigam, A. H. Van GennipAbstract:The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to β-alanine and β-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and Dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-β-amino aciduria, due to a deficiency of β-ureidopropionase, was easily diagnosed at the metabolite level using HPLC–tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of β-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of β-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and Dihydropyrimidinase was present. Thus, HPLC–tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of β-ureidopropionase.
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Dihydropyrimidinase deficiency confirmation of the enzyme defect in dihydropyrimidinuria
Journal of Inherited Metabolic Disease, 1997Co-Authors: A. H. Van Gennip, R. A. De Abreu, Jan J. Rotteveel, P Vreken, G H Van Lenthe, J A J M Bakkeren, A B P Van KuilenburgAbstract:Dihydropyrimidinase (DHP, EC 3.5.2.2) is the second enzyme in the degradation pathway of uracil and thymine. It catalyses the degradation of both dihydrouracil and dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. So far, four cases of dihydropyrimidinuria (McKusick 222748) have been reported (Duran et al 1991; Henderson et al 1993; Bakkeren et al, personal communication, 1994; Ohba et al 1994). The patients show a variable clinical phenotype comprising seizures or epileptic attacks (3 out of 4 patients) mental retardation (2 patients), growth retardation (1 patient) and dysmorphic features (1 patient). Since these patients excrete large amounts of dihydrouracil and dihydrothymine and moderate amounts of uracil and thymine in their urine, they can easily be detected (Van Gennip et al 1993). On the basis of the characteristic urinary metabolite profile it is assumed that the disease is caused by a deficiency of DHP. The direct measurement of the activity of DHP in patients has been hampered by the fact that the enzyme is almost exclusively expressed in liver tissue. Here, we provide for the first time direct evidence at the enzyme level for a deficient activity of DHP in liver in a patient with dihydropyrimidinuria.
Nanaya Tamaki - One of the best experts on this subject based on the ideXlab platform.
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Dihydropyrimidinase deficiency and severe 5 fluorouracil toxicity
Clinical Cancer Research, 2003Co-Authors: Andre B P Van Kuilenburg, Rutger Meinsma, Koichi Matsuda, Nanaya Tamaki, Bernard A Zonnenberg, Lida Zoetekouw, Frank Baas, A. H. Van GennipAbstract:Dihydropyrimidinase (DHP) is the second enzyme in the catabolism of 5-fluorouracil (5FU), and it has been suggested that patients with a deficiency of this enzyme are at risk from developing severe 5FU-associated toxicity. In this study, we demonstrated for the first time that in one patient the severe toxicity, after a treatment with 5FU, was attributable to a partial deficiency of DHP. Analysis of the DHP gene showed that the patient was heterozygous for the missense mutation 833G>A (G278D) in exon 5. Heterologous expression of the mutant enzyme in Escherichia coli showed that the G278D mutation leads to a mutant DHP enzyme without residual activity. An analysis for the presence of this mutation in 96 unrelated Dutch Caucasians indicates that the allele frequency in the normal population is <0.5%. Our results show that a partial DHP deficiency is a novel pharmacogenetic disorder associated with severe 5FU toxicity.
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molecular cloning and sequencing of a cdna encoding Dihydropyrimidinase from the rat liver
Biochimica et Biophysica Acta, 1996Co-Authors: Koichi Matsuda, Naoki Hamajima, Makoto Sasaki, Masaru Nonaka, Masae Kaneko, Shigeko Fujimoto Sakata, Nanaya TamakiAbstract:Abstract A cDNA encoding Dihydropyrimidinase has been isolated from a rat cDNA library. The N-terminal and an internal amino acid sequences were determined, and PCR primers were designed based on these sequences. Using a cDNA fragment amplified by RT-PCR with these primers, three cDNA clones were isolated from a rat liver library. The clone with the longest insert of 2129 bp contained a 1557 bp open reading frame encoding a polypeptide of 519 residues with a molecular mass of 56 832 Da.
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analysis of cyclic feed intake in rats fed on a zinc deficient diet and the level of Dihydropyrimidinase ec 3 5 2 2
British Journal of Nutrition, 1995Co-Authors: Nanaya Tamaki, Shigeko Fujimotosakata, Mariko Kikugawa, Masae Kaneko, Satomi Onosaka, Tatsuya TakagiAbstract:The body weight and feed intake of rats fed on a Zn-deficient diet for 28 d were reduced compared with those of control rats. The feed intakes of the Zn-deficient and control groups during the period were 10·2 (SE 0·3) and 15·7 (SE 0·2) g/d respectively. Cyclic variations in feed intake and body-weight changes were found in analysis not only of all the data for five rats but also that in each individual rat. Cosinor analysis revealed that the cyclical period of both the feed intake and body-weight change in the Zn-deficient rats was 3·5 (SE 0·1) d. The mesor and amplitude value of the feed intake in the Zn-deficient rats was 10 1 (SE 0·4) g/d and 3·5 (SE 0·5) g/d respectively, and that of body-weight change was 1·4 (SE 0·1) g/d and 7·9 (SE 1·3) gObihiro d respectively. Among pyrimidine-catabolizing enzymes, Dihydropyrimidinase (EC3.5.2.2) activity showed significantretardation in the Zn-deficient rat liver with decrease of the enzyme protein. The ratio of apo-form to holo-form Dihydropyrimidinase in the liver was not affected by the Zn-deficient diet.
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purification characterization and inhibition of Dihydropyrimidinase from rat liver
FEBS Journal, 1994Co-Authors: Mariko Kikugawa, Shigeko Fujimotosakata, Masae Kaneko, Tatsuya Takagi, Mitsuko Maeda, Koichi Kawasaki, Nanaya TamakiAbstract:Dihydropyrimidinase (DHPase) was purified 564-fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE-Sepharose CL-6B, carboxymethyl-Sepharose CL-6B, hydroxyapatite and Sephacryl S-300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6-dihydrouracil (H2Ura) to N-carbamoyl-β-alanine or 5,6-dihydrothymine (H2Thy) to N-carbamoyl-β-aminoisobutyric acid. Authentic 5-bromo-5,6-dihydrouracil (BrH2Ura) and commercially available H2Thy were racemic. However, these 5-substituted 5,6-dihydropyrimidines were hydrolyzed by over 96% and 98%, respectively, by DHPase. These results suggest that Dihydropyrimidinase has no stereo specificities for 5-substituents of H2Ura. The addition of H2Ura and H2Thy competitively inhibited the enzyme activity against BrH2Ura. However, the addition of N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid showed hyperbolic mixed-type inhibition, when BrH2Ura was used as the substrate. The values of the dissociation constants of BrH2Ura, N-carbamoyl-β-alanine and N-carbamoyl-β-aminoisobutyric acid were 17 μM, 0.38 mM and 0.38 mM, respectively. DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atom/subunit. Zn2+ also inhibited the hydrolysis of BrH2Ura by the enzyme. The Ki for Zn2+ as an inhibitor of DHPase was 23 μM, and the maximum rate of inactivation was 0.057 min−1 at 37°C. H2Ura and H2Thy protected the enzyme activity from Zn2+ inactivation.