Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Pyrimidine ribonucleoside catabolic enzyme activities ofPseudomonas pickettii

Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogenPseudomonas pickettii were examined. Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth. Thin-layer chromatographic separation of the uridine and cytidine catabolities produced byP. pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity. Its presence was confirmed by enzyme assay. Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells. Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source. Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH₄)₂SO₄ served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity. Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source. Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable inP. pickettii. The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate. Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.     

Pyrimidine base catabolism in Pseudomonas putida biotype B

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl--alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.     

Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

A dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis ATCC 17414 was isolated and characterized in this study. Initially, reductive catabolism of uracil was confirmed to be active in ATCC 17414 cells. Following chemical mutagenesis and d-cycloserine counterselection, a mutant strain unable to utilize uracil as a nitrogen source was identified. It was also unable to utilize thymine as a nitrogen source but could use either dihydrouracil or dihydrothymine as a sole source of nitrogen. Subsequently, it was determined that the mutant strain was deficient for the initial enzyme in the reductive pathway dihydropyrimidine dehydrogenase. The lack of dehydrogenase activity did not seem to have an adverse effect upon the activity of the second reductive pathway enzyme dihydropyrimidinase activity. It was shown that both dihydropyrimidine dehydrogenase and dihydropyrimidinase levels were affected by the nitrogen source present in the growth medium. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were elevated after growth on uracil, thymine, dihydrouracil or dihydrothymine as a source of nitrogen.     

Reductive catabolism of uracil and thymine by Burkholderia cepacia

Catabolism of uracil and thymine in Burkholderia cepacia ATCC 25416 was shown to occur using a reductive pathway. The first pathway enzyme, dihydropyrimidine dehydrogenase, was shown to utilize NADPH as its nicotinamide cofactor. Growth of B. cepacia on pyrimidine bases as the nitrogen source instead of on ammonium sulfate increased dehydrogenase activity at least 32-fold. The second and third reductive pathway enzymes, dihydropyrimidinase and N-carbamoyl-β-alanine amidohydrolase, respectively, exhibited activities elevated more than 21-fold when pyrimidine or dihydropyrimidine bases served as the nitrogen source rather than ammonium sulfate. The pathway enzyme activities were induced after growth on 5-methylcytosine. Received: 17 January 1997 / Accepted: 5 May 1997     

Pyrimidine catabolism in Pseudomonas aeruginosa

Pyrimidine catabolism in Pseudomonas aeruginosa was investigated. It was found that the pyrimidine bases uracil and thymidine as well as their respective reductive catabolic products could be utilized as sole sources of nitrogen. Reductive degradation of the pyrimidine bases was noted. The reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase were all detected in minimal medium grown cells. Induction of pyrimidine catabolism by uracil was observed in this pseudomonad. Pyrimidine degradation in P. aeruginosa was not subject to catabolite repression.     

Activity of pyrimidine degradation enzymes in normal tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and beta-ureidopropionase (beta-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of beta-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and beta-UP were also present in a variety of other tissues, especially in bronchus.     

Activity of Pyrimidine Degradation Enzymes in Normal Tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and ß-ureidopropionase (ß-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of ß-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and ß-UP were also present in a variety of other tissues, especially in bronchus.     

Analysis of pyrimidine catabolism in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and beta-alanine metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.     

Isolation and characterization of an Escherichia coli B mutant strain defective in uracil catabolism

A reductive pathway of uracil catabolism was shown to be functioning in Escherichia coli B ATCC 11303 by virtue of thin-layer chromatographic and enzyme analyses. A mutant defective in uracil catabolism was isolated from this strain and subsequently characterized. The three enzyme activities associated with the reductive pathway of pyrimidine catabolism were detectable in the wild-type E. coli B cells, while the mutant strain was found to be deficient for dihydropyrimidine dehydrogenase activity. The dehydrogenase was shown to utilize NADPH as its nicotinamide cofactor. Growth of ATCC 11303 cells on uracil or glutamic acid instead of ammonium sulfate as a nitrogen source increased the reductive pathway enzyme activities. The mutant strain exhibited increased catabolic enzyme activities after growth on ammonium sulfate or glutamic acid.     

Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase

Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.     

Evaluation of pyrimidine- and hydantoin-degrading enzyme activities in aerobic bacteria

Abstract The enzyme activities responsible for the reductive pyrimidine base degradation by aerobic bacteria, which produce hydantoin-degrading enzymes, were investigated. Pseudomonas putida IFO 12996, which is a d-stereospecific hydantoinase producer, has dihydropyrimidinase activity, and Comamonas sp. E222c and Blastobacter sp. A17p-4, which are N-carbamoyl-D-amino acid amidohydrolase producers, have β-ureidopropionase activity. Blastobacter sp. also possesses both d-stereospecific hydantoinase and dihydropyrimidinase activities. Thus, two amide ring-opening activities and/or two N -carbamoyl amino acid-hydrolyzing activities coexist in these bacteria. However, the differences of the induction levels of each enzyme activities for the several pyrimidine- and hydantoin-related compounds suggest that these corresponding amide ring-opening or N -carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.     

Pyrimidine catabolism: individual characterization of the three sequential enzymes with a new assay

We have developed a one-dimensional thin-layer chromatography procedure that resolves the initial substrate uracil and its catabolic derivatives dihydrouracil, N-carbamoyl-beta-alanine (NCBA) and beta-alanine. This separation scheme also simplifies the preparation of the radioisotopes of N-carbamoyl-beta-alanine and dihydrouracil. Combined, these methods make it possible to assay easily and unambiguously, jointly or individually, all three enzyme activities of uracil catabolism: dihydropyrimidine dehydrogenase, dihydropyrimidinase, and N-carbamoyl-beta-alanine amidohydrolase. Earlier reports had presented data suggesting that these three enzyme activities were combined in a complex because they appeared to be controlled at a single genetic locus [Dagg, C. P., Coleman, D.L., & Fraser, G.M. (1964) Genetics 49, 979-989] and because they appeared able to channel metabolites [Barrett, H.W., Munavalli, S.N., & Newmark, P. (1964) Biochim. Biophys. Acta 91, 199-204]. Although the three enzymes from rat liver have similar sizes, with apparent molecular weights of 218 000 for dihydropyrimidine dehydrogenase, 226 000 for dihydropyrimidinase, and 234 000 for NC beta A amidohydrolase, they are easily separated from each other. Kinetic studies show no evidence of substrate channeling and therefore do not support a model for an enzyme complex. The earlier reports may be explained by our studies on the amidohydrolase, which suggest that under certain conditions this enzyme may become the rate-limiting step in uracil catabolism.     

The RihA, RihB, and RihC ribonucleoside hydrolases of Escherichia coli. Substrate specificity, gene expression, and regulation

Pyrimidine-requiring cdd mutants of Escherichia coli deficient in cytidine deaminase utilize cytidine as a pyrimidine source by an alternative pathway. This has been presumed to involve phosphorylation of cytidine to CMP by cytidine/uridine kinase and subsequent hydrolysis of CMP to cytosine and ribose 5-phosphate by a putative CMP hydrolase. Here we show that cytidine, in cdd strains, is converted directly to cytosine and ribose by a ribonucleoside hydrolase encoded by the previously uncharacterized gene ybeK, which we have renamed rihA. The RihA enzyme is homologous to the products of two unlinked genes, yeiK and yaaF, which have been renamed rihB and rihC, respectively. The RihB enzyme was shown to be a pyrimidine-specific ribonucleoside hydrolase like RihA, whereas RihC hydrolyzed both pyrimidine and purine ribonucleosides. The physiological function of the ribonucleoside hydrolases in wild-type E. coli strains is enigmatic, as their activities are paralleled by the phosphorolytic activities of the nucleoside phosphorylases, and a triple mutant lacking all three hydrolytic activities grew normally. Furthermore, enzyme assays and lacZ gene fusion analysis indicated that rihB was essentially silent unless activated by mutation, whereas rihA and rihC were poorly expressed in glucose medium due to catabolite repression.     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

On the iron-sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase.

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.     

The presence of 11-methyloctadec-11-enoic acid in the extractable lipids of Pseudomonas vesicularis

Abstract The polar lipids of Pseudomonas vesicularis do not contain a methyleneoctadecanoic acid but instead the isomeric 11-methyloctadec-11-enoic acid. The presence of the latter acid distinguishes the lipids of P. vesicularis from those of P. diminuta , but links them with the lipids of Rhizobium and Caulobacter species.     

Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency

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.