Publications

• Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination.

  Lohkamp B., Voevodskaya N., Lindqvist Y., Dobritzsch D.

  In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. Th ... >> More

  In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding. << Less

  Biochim. Biophys. Acta 1804:2198-2206(2010) [PubMed] [EuropePMC]

• A functional analysis of the pyrimidine catabolic pathway in Arabidopsis.

  Zrenner R., Riegler H., Marquard C.R., Lange P.R., Geserick C., Bartosz C.E., Chen C.T., Slocum R.D.

  * Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to beta-alanine, CO(2) and NH(3) through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and beta-ureidopropionase (EC 3. ... >> More

  * Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to beta-alanine, CO(2) and NH(3) through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and beta-ureidopropionase (EC 3.5.1.6, PYD3). * A proposed function of this pathway, in addition to the maintenance of pyrimidine homeostasis, is the recycling of pyrimidine nitrogen to general nitrogen metabolism. PYD expression and catabolism of [2-(14)C]-uracil are markedly elevated in response to nitrogen limitation in plants, which can utilize uracil as a nitrogen source. * PYD1, PYD2 and PYD3 knockout mutants were used for functional analysis of this pathway in Arabidopsis. pyd mutants exhibited no obvious phenotype under optimal growing conditions. pyd2 and pyd3 mutants were unable to catabolize [2-(14)C]-uracil or to grow on uracil as the sole nitrogen source. By contrast, catabolism of uracil was reduced by only 40% in pyd1 mutants, and pyd1 seedlings grew nearly as well as wild-type seedlings with a uracil nitrogen source. These results confirm PYD1 function and suggest the possible existence of another, as yet unknown, activity for uracil degradation to dihydrouracil in this plant. * The localization of PYD-green fluorescent protein fusions in the plastid (PYD1), secretory system (PYD2) and cytosol (PYD3) suggests potentially complex metabolic regulation. << Less

  New Phytol. 183:117-132(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Acid base catalytic mechanism of the dihydropyrimidine dehydrogenase from pH studies.

  Podschun B., Jahnke K., Schnackerz K.D., Cook P.F.

  Primary deuterium (NADPH(D)), solvent deuterium, and multiple isotope effects and the pH dependence of kinetic parameters have been used to probe the mechanism of the dihydropyrimidine dehydrogenase from pig liver. Isotope effect and pH-rate data suggest a rate-determining reductive half-reaction ... >> More

  Primary deuterium (NADPH(D)), solvent deuterium, and multiple isotope effects and the pH dependence of kinetic parameters have been used to probe the mechanism of the dihydropyrimidine dehydrogenase from pig liver. Isotope effect and pH-rate data suggest a rate-determining reductive half-reaction in which reduction of the flavin by NADPH has only a minor rate limitation (DV approximately D(V/KNADPH) approximately 1.1), while protonation of the flavin at N-1 occurring in a step following reduction is slow (D2OV = 3, while D2O(V/KNADPH) = 2). An enzymatic general acid with a pK of 8.2 is required to protonate N-1 of the flavin. In the second half-reaction, uracil is reduced at C-6 by flavin and protonated on the opposite face at C-5 by an enzymatic general acid with a pK of 9. The hydride transfer from N-5 of the flavin to C-5 of uracil is facilitated by an enzymatic general base with a pK of 5.6 that accepts a proton from N-1 of the flavin. There is also evidence from the pH dependence of V and the V/K for reduced dinucleotide substrates that a second enzyme residue with a pK of 6.4 must be unprotonated for optimum activity, but is not essential for activity. None of the functional groups reflected in the V/KNADPH pH-rate profile have a role in binding, while both of those observed in the V/Kuracil profile have a role in binding as shown by the pH dependence of the dissociation constants for the competitive inhibitors ATP-ribose and 2,6-dihydroxypyridine. << Less

  J Biol Chem 268:3407-3413(1993) [PubMed] [EuropePMC]

• Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH.

  Porter D.J., Spector T.

  Steady-state and pre-steady-state kinetic data were used to determine the kinetic mechanism for bovine liver dihydropyrimidine dehydrogenase (DPDase). Steady-state kinetic data suggested a random rapid-equilibrium mechanism with Km values for NADPH and uracil of 0.12 microM and 0.8 microM, respect ... >> More

  Steady-state and pre-steady-state kinetic data were used to determine the kinetic mechanism for bovine liver dihydropyrimidine dehydrogenase (DPDase). Steady-state kinetic data suggested a random rapid-equilibrium mechanism with Km values for NADPH and uracil of 0.12 microM and 0.8 microM, respectively, and a kcat of 1.6 s-1 in Tris buffer at pH 8.0 and 37 degrees C. The dissociation constant of DPDase for NADPH at 25 degrees C in the absence of uracil (0.09 microM) was similar to the Km for NADPH. DPDase also catalyzed the exchange of tritium in [4S-3H,4R-1H]NADP3H with solvent protons in the absence of uracil. DPDase inactivated by 5-ethynyluracil, which covalently modifies the enzyme at the uracil binding site, catalyzed the exchange reaction at the same rate (1 s-1) as native enzyme. Thus, the interaction of NADPH with DPDase was independent of the uracil binding site. Because DPDase catalyzed the exchange of deuterium in [4S-2H,4R-1H]NADP2H with solvent protons with a rate constant of 5.4 s-1, which was significantly larger than that for tritium, the analogous rate constant for exchange of the 4-hydrogen in NADPH must be significantly larger than 5 s-1. Consequently, intermediates on the exchange pathway were kinetically competent to participate in the reduction of uracil by NADPH (kcat = 1.6 s-1). Rate constants for reduction of DPDase by NADPH and 5,6-dihydrouracil were several orders of magnitude greater than kcat. The rate constants for dissociation of E.NADP+ (15 s-1) and for dissociation of E.5,6-dihydrouracil (> 250 s-1) were also greater than kcat. These results supported a random rapid-equilibrium kinetic mechanism and suggested kcat was an internal electron transfer between enzymic prosthetic groups. << Less

  J Biol Chem 268:19321-19327(1993) [PubMed] [EuropePMC]

• Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus.

  Schmitt U., Jahnke K., Rosenbaum K., Cook P.F., Schnackerz K.D.

  Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The enzyme is a homotetramer with a subunit molecular mass of 52 kDa. The absorption spectrum ... >> More

  Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The enzyme is a homotetramer with a subunit molecular mass of 52 kDa. The absorption spectrum of the bacterial dihydropyrimidine dehydrogenase has maxima in the 300- and 400-nm region, suggesting a flavoprotein. The enzyme contains 4 mol FMN, about 24 mol iron and acidlabile sulfide per mole of protein, implying a flavoprotein with FeS centers. The bacterial dehydrogenase is NADPH dependent with B-side stereospecificity. The initial velocity patterns of the bacterial dehydrogenase together with isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium random kinetic mechanism, which is in contrast to results obtained for dihydropyrimidine dehydrogenase from pig liver. The pig liver enzyme adheres to a nonclassical two-site ping-pong kinetic mechanism [B. Podschun, P. F. Cook, and K. D. Schnackerz (1990) J. Biol. Chem. 265, 12966-12972], whereas for the bovine enzyme a rapid equilibrium random kinetic mechanism was proposed based on steady-state kinetic data [D. J. T. Porter and T. Spector (1993) J. Biol. Chem. 268, 19321-19327]. << Less

  Arch Biochem Biophys 332:175-182(1996) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of dihydrothymine dehydrogenase from rat liver.

  Shiotani T., Weber G.

  Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel electrophoresis, sedimentation velocity, and Ultrogel ACA-34 elution profile. The enzyme has a molecular weight of 220,000 +/-5,00 ... >> More

  Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel electrophoresis, sedimentation velocity, and Ultrogel ACA-34 elution profile. The enzyme has a molecular weight of 220,000 +/-5,000 as determined by Ultrogel ACA-34 and sedimentation equilibrium The s20,w value of the enzyme was 9.2 S. The isoelectric point was at pH 5.25. The enzyme is composed of two identical subunits of an approximate molecular weight of 110,000 +/-3,000 as determined by sodium dodecyl sulfate disc gel electrophoresis. The enzyme contains 4 mol of FAD and 3 mol of iron per mol of enzyme. Flavin released from the enzyme by boiling was identified as FAD by absorption spectra and thin layer chromatography, indicating that the enzyme is a flavometal protein. During dialysis, the enzyme was stabilized by 2-mercaptoethanol, but neither NADPH nor thymine was effective. The relative rates of reduction of pyrimidine analogues substituted at position 5 were 5-fluorouracil > 5-bromouracil > 5-diazouracil > 5-iodouracil > 5-nitrouracil, with 5-fluorouracil and 5-diazouracil 70% faster than thymine. Uracil was reduced 25% faster than thymine. The pH optimum for the forward and reverse reactions was 7.4. In the presence of NADPH, the apparent Km was 2.6 microM for thymine and 1.8 microM for uracil. Apparent Km for NADPH was 15 microM with thymine as substrate and 11 microM with uracil. In the reverse reaction, apparent Km values were 43 microM for dihydrothymine and 193 microM for dihydrouracil; apparent Km for NADP+ was 3.8 microM with dihydrothymine as substrate and 2.9 microM with dihydrouracil. << Less

  J Biol Chem 256:219-224(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Porcine recombinant dihydropyrimidine dehydrogenase: comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes.

  Rosenbaum K., Jahnke K., Curti B., Hagen W.R., Schnackerz K.D., Vanoni M.A.

  Biochemistry 37:17598-17609(1998) [PubMed] [EuropePMC]

• Stereochemistry of NADPH oxidation by dihydropyrimidine dehydrogenase from pig liver.

  Podschun B.

  Dihydropyrimidine dehydrogenase reduces uracil to 5,6-dihydrouracil in a strictly NADPH-dependent reaction. Either by analysing the 1H-NMR spectra of the NADP+ products formed or by determination of the kinetic isotope effects of stereospecifically deuterated coenzymes dihydropyrimidine dehydrogen ... >> More

  Dihydropyrimidine dehydrogenase reduces uracil to 5,6-dihydrouracil in a strictly NADPH-dependent reaction. Either by analysing the 1H-NMR spectra of the NADP+ products formed or by determination of the kinetic isotope effects of stereospecifically deuterated coenzymes dihydropyrimidine dehydrogenase was found to abstract specifically the pro-S hydrogen of NADPH, making it a member of the B-side stereospecific class of dehydrogenases. << Less

  Biochem Biophys Res Commun 182:609-616(1992) [PubMed] [EuropePMC]

• Kinetic mechanism of dihydropyrimidine dehydrogenase from pig liver.

  Podschun B., Cook P.F., Schnackerz K.D.

  Data on initial velocity and isotope exchange at equilibrium suggest a nonclassical ping-pong mechanism for the dihydropyrimidine dehydrogenase from pig liver. Initial velocity patterns in the absence of inhibitors appeared parallel at low reactant concentration, with substrate inhibition by NADPH ... >> More

  Data on initial velocity and isotope exchange at equilibrium suggest a nonclassical ping-pong mechanism for the dihydropyrimidine dehydrogenase from pig liver. Initial velocity patterns in the absence of inhibitors appeared parallel at low reactant concentration, with substrate inhibition by NADPH that is competitive with uracil and with substrate inhibition by uracil that is uncompetitive with NADPH. The Km values for both uracil (1 microM) and NADPH (7 microM) are low. As a result, it was difficult to determine whether the initial velocity pattern in the absence of added inhibitors was parallel. Thus, the pattern was redetermined in the presence of the dead-end inhibitor 2,6-dihydroxypyridine, which binds to both sites. This treatment effectively eliminates the inhibition by both substrates and increases their Km values, giving a strictly parallel pattern. Product and dead-end inhibition patterns are consistent with a mechanism in which NADPH reduces the enzyme at site 1 and electrons are transferred to site 2 to reduce uracil to dihydrouracil. The predicted mechanism is corroborated by exchange between [14C] NADP and NADPH as well as [14C]thymine and dihydrothymine in the absence of the other substrate-product pair. << Less

  J Biol Chem 265:12966-12972(1990) [PubMed] [EuropePMC]