Claus Wasternack

Pyrimidine-degrading enzymes. Purification and properties of β-ureidopropionase of Euglena gracilis

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, were detected in a crude extract. beta-Ureidopropionase (N-carbamoyl-beta-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of beta-ureidopropionase for beta-ureidopropionate 3.8 . 10(-5) M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60 degrees C, stimulation by Mg2+, inhibition by Cu2+, Mr approximately 1.5--2 . 10(6). beta-Ureidoisobutyrate, the intermediate of thymine degradation, and beta-ureidopropionate are competing substrates of beta-ureidopropionase (Ki = Km of beta-ureidopropionase for beta-ureidoisobutyrate 1.8 . 10(-5) M). Structural analogues of beta-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of beta-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of beta-ureidopropionase in pyrimidine degradation.

Degradation of Pyrimidines — Enzymes, Localization and Role in Metabolism

Summary Pyrimidines can be degraded (1), reductively; (2), oxydatively by attack of oxygen in the position 5 or 6 of the pyrimidine ring; (3), by oxydative demethylation of thymine; (4), by decarboxylation of orotic acid to uracil; (5), by reductive degradation of orotic acid. The reductive degradation is the prevailing degrading process in nature. It is catalyzed by uracil reductase, dihydropyrimidinase and β-ureidopropionase. Properties and location of enzymes are described. Distribution and significance of pyrimidine degradation in different organisms are presented. Its presence in animals is preferentially discussed with respect to correlation between anabolic and catabolic reactions in terms of the “Molecular Correlation Concept”. Some plants use reverse reactions of pyrimidine degradation for biosynthesis of some pyrimidine-containing secondary products.

Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance α-AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and α-AIB is inhibited by cycloheximide in a different manner after 5–10 min.

Molecular analysis of mitochondrial DNA from tomato cell suspension cultures

SummaryMitochondrial DNA (mtDNA) from Lycopersicon esculentum was purified from cell suspension cultures. The DNA, isolated from mitochondria purified by two successive sucrose density gradients, was uncontaminated with nuclear DNA or DNA from proplastids. The total molecular weights of BamHI, BglI, and BglII fragments indicate a mitochondrial genome size of at least 270 kb. Cross hybridization between tomato mtDNA and cloned spinach plastid genes revealed some homology. In hybridization experiments using cloned mitochondrial rRNA genes and BamHI digested total mtDNA the presence of recombination repeats is demonstrated.

Subcellular localization of some purine-metabolizing enzymes in tomato (Lycopersicon esculentum) cells grown in suspension

Summary Cell-free extracts from cells of Lycopersicon esculentum were assayed for the presence of activities of 12 enzymes involved in purine metabolism. Only four of them, 5′-nucleotide phosphatase (EC 3.1.3.5), adenine phosphoribosyltransferase (EC 2.4.2.7), 5′-methylthioadenosine hydrolase (EC 3.2.2.9), and S-adenosylhomocysteine hydrolase (EC 3.3.1.1) occurred at levels which allowed their intracellular localization. This was performed by differential centrifugation, by sucrose density gradient centrifugation, by controlled lysis of protoplasts, or by isolation of vacuoles. All the enzymes mentioned were found to be located in the cytosol.

Degradation of pyrimidines in Euglena gracills III. Ratio of uracil to thymine degradation

In photoorganotrophically-grown cells of Euglena gracilis var. Z uracil (Ura) and thymine (Thy) were degraded in a fixed ratio of 1 : 4. The predominant degradation of Thy can be measured in vivo by the kinetics of Thy and Ura degradation, by the effect of catabolic intermediates at different concentrations and by double labelling experiments with [5−3H]Ura and [2−14C]Thy. In the latter type of experiment, 3H/14C of isolated dihydrouracil (DHUra) and dihydrothymine (DHThy) is respectively 110 of the starting promortion of precursors, suggesting a predominant degradation of Thy. Enhanced anabolic reactions of pyrimidine compounds can be produced by inhibition of degradation with dihydropyrimidines.

Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members

Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles.

Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.

Localization of Carbamyl Phosphate-metabolizing Enzymes in Different Organelles within Tomato (Lycopersicon esculentum) Cells

Summary In plant cells carbamyl phosphate is provided by only one carbamyl phosphate synthetase for the pyrimidine biosynthesis as well as for the arginine synthesis. Using a cytochemical technique to detect the in situ precipitation of orthophosphate ions liberated by the aspartate transcarbamylase (ATCase) or by the ornithine transcarbamylase (OTCase) reaction, respectively, we could show that the two enzymes utilizing carbamyl phosphate are localized in different organelles within cultured tomato cells. Whereas ATCase is localized inside the nuclei, OTCase is confined to the vacoule. This enzyme location is discussed in relation to results on other plant cells, emphasizing the advantage of the cytochemical approach used.

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

Abstract 1. 1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase† was studied in a cell suspension culture of tomato ( Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis . 2. 2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH ( E. gracilis ). 3. 3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients. 4. 4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients. 5. 5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase. 6. 6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded. 7. 7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.