Publications

• Bovine liver dihydropyrimidine amidohydrolase: purification, properties, and characterization as a zinc metalloenzyme.

  Brooks K.P., Jones E.A., Kim B.D., Sander E.G.

  Beef liver dihydropyrimidine amidohydrolase has been purified to homogeneity using both an electrophoretic and a hydrophobic chromatographic method. The enzyme is a tetramer with a molecular weight of 226,000 g mol-1, a subunit molecular weight of 56,500 g mol-1, and contains 4 mol of tightly boun ... >> More

  Beef liver dihydropyrimidine amidohydrolase has been purified to homogeneity using both an electrophoretic and a hydrophobic chromatographic method. The enzyme is a tetramer with a molecular weight of 226,000 g mol-1, a subunit molecular weight of 56,500 g mol-1, and contains 4 mol of tightly bound (Ks greater than or equal to 1.33 X 10(9) M-1) Zn2+ per mole of active enzyme. The enzyme appears to be a true Zn2+ metalloenzyme because there exists a direct proportionality between enrichment of Zn2+ and active enzyme during purification, there is an almost quantitative relationship between the loss of both enzyme activity and Zn2+ during 8-hydroxyquinoline-5-sulfonic acid treatment to form apoenzyme, Zn2+ and Co2+ reactivate dipicolinic acid-inhibited enzyme, and saturating concentrations of a substrate, dihydrothymine, protect against 8-hydroxyquinoline-5-sulfonic acid inhibition. EDTA does not inhibit the enzyme; however, 8-hydroxyquinoline-5-sulfonic acid, o-phenanthroline, and 2,6-dipicolinic acid cause a time-dependent loss in activity which follows pseudo-first-order kinetics. Analysis of the resulting kinetic data for these three chelators indicates that the reaction pathway involves the formation of an enzyme-Zn2+-chelator ternary complex which then dissociates to form apoenzyme and a Zn2+-chelator complex. Like other Zn2+ metalloenzymes, the enzyme is inhibited by a number of substituted sulfonamides. In the case of p-nitrobenzenesulfonamide, this inhibition is competitive in nature. Using the purified enzyme, kinetic constants were determined for a variety of dihydropyrimidines, ureidocarboxylic acids, and hydantoin substrates. Normal hyperbolic kinetics were observed for the hydrolysis of the cyclic compounds, but the cyclization of the ureidoacids showed biphasic kinetics and different values of Km can be estimated at either high or low concentrations of these substrates. << Less

  Arch Biochem Biophys 226:469-483(1983) [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.

• Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties.

  Gojkovic Z., Rislund L., Andersen B., Sandrini M.P., Cook P.F., Schnackerz K.D., Piskur J.

  Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and s ... >> More

  Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-beta-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones. << Less

  Nucleic Acids Res. 31:1683-1692(2003) [PubMed] [EuropePMC]

• PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway.

  Gojkovic Z., Jahnke K., Schnackerz K.D., Piskur J.

  Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular techniq ... >> More

  Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast. The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complementing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases. The organisation of PYD2 showed a couple of specific features. The 542-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, allantoinases and uricases from various organisms. Surprisingly, the URA4 gene from S. cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several other non-fungal organisms. The S. kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely consisting of similar if not identical subunits each with a molecular mass of 59 kDa. The S. kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase. On the other hand, the regulation of the yeast PYD2 gene and DHPase seem to be different from that in other organisms. DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil. Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S. kluyveri. << Less

  J. Mol. Biol. 295:1073-1087(2000) [PubMed] [EuropePMC]