UMP Pyrophosphorylase Research Articles

UMP Pyrophosphorylase: A Moonlighting Protein with Essential Functions in Chloroplast Development and Photosynthesis Establishment.

Pyrimidine nucleotides (e.g. deoxy-CTP, deoxy-TTP, UTP, and CTP) are essential building blocks for DNA and RNA synthesis in all organisms. In plants, Suc and cell wall polymer synthesis depend on pyrimidine nucleotide-derived substrates, such as UDP-Glc, and important classes of membrane lipids are

19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism

Flucytosine (5-FC)-resistant strains were isolated from the haploid opportunistic pathogen Candida glabrata by UV-induced mutation and fluoropyrimidine selection. These strains were characterized biochemically, and the metabolism of fluorinated pyrimidines was studied by 19F nuclear magnetic resonance spectroscopy. No evidence was obtained from these studies for degradative metabolism of the fluorinated derivatives. In the parental susceptible strain of C. glabrata, 5-fluorouracil but not 5-FC was detected within the cells. 5-Fluorouracil was also present in the culture supernatant after incubation of the cells with 5-FC. The distribution of fluorinated derivatives within the 5-FC-resistant strains was consistent with their genotype. Two strains of C. glabrata which had only a partial loss of cytosine deaminase and UMP pyrophosphorylase activity had high levels of resistance to 5-FC. Both C. glabrata and Candida albicans were susceptible to 5-fluorouridine. This compound but not the anticancer drug 5-fluoro-2-deoxyuridine was shown to be transported into susceptible cells by a specific uridine permease.

Conditions for Mutagenesis of the Nitrogen-fixing Cyanobacterium Anabaena variabilis

Chemically induced mutation in the cyanobacterium Anabaena variabilis was studied using resistance to the pyrimidine analogue 5′-fluorocytosine as a genetic marker which can be selected positively. Cytosine is metabolized through uracil and the UMP pyrophosphorylase ‘salvage’ pathway in this photoautotroph, as it is in enteric bacteria. Treatment with various concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) gave the highest frequencies of 5FC-resistant mutants when lethality approximated 99%, irrespective of the exposure time or mutagen concentration. The pH of the incubation medium strongly influenced mutation; exposure to MNNG at pH 6·0 yielded 13-fold higher frequencies of mutants than at pH 7·5. The greatest frequency of resistant cells was found after cultures had undergone six or more doublings following mutagenesis. The mutation frequencies obtained by treatment with MNNG were approximately 4- and 25-fold higher than those after exposure under empirically defined conditions to diethyl sulphate or nitrous acid, respectively, and 1·4 × 103-fold higher than the frequency of spontaneous mutation. Neither chloramphenicol-inhibited nor caffeine-sensitive systems capable of repairing MNNG-induced DNA damage were observed.

The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans.

In terms of genetically determined susceptibility to the clinical antifungal agent 5-fluorocytosine (5-FC), Candida albicans may be homozygous sensitive (FCY/FCY), homozygous resistant (fcy/fcy), or heterozygous (fcy/FCY). Although heterozygotes are only slightly resistant, they occur at significant frequency among clinical strains and carry preexisting resistance determinants which may be responsible, following homozygosis, for treatment failures. There are two resistance genes (FCY1 and FCY2) known. Resistance in fcy1/fcy1 strains was associated with decreased UMP pyrophosphorylase activity, whereas resistance in fcy2/fcy2 strains was associated with decreased cytosine deaminase activity. These results were confirmed and extended in a 19F nuclear magnetic resonance study of 5-FC uptake and metabolism in genetically defined strains. By means of hybridization via spheroplast fusion, a complementation test was devised to test allelism of resistance determinants. Resistance to 5-FC was employed as a useful genetic marker in basic studies. In tetraploid hybrids which bore appropriate fcy markers, it was possible to select for reduction in ploidy by selecting for increased resistance to 5-FC; a novel parasexual system was thus generated (2n x 2n----4n----2n). In linkage studies, the gene FCY1 was shown to be linked to the gene HIS. Reciprocal mitotic recombination was demonstrated repeatedly with fcy1 and his alleles in cis and in trans configurations and evidence for nonreciprocal recombination (mitotic gene conversion) was also obtained. In Cryptococcus neoformans, mutation in either of two genes (FCY1, FCY2) is sufficient to confer resistance. These genes behave as simple Mendelian determinants which recombine freely. Diploid C. neoformans heterozygous for resistance (FCY/fcy) provided useful strains in which to develop genetic mapping methodology based on mitotic recombination.

Noninvasive and quantitative 19F nuclear magnetic resonance study of flucytosine metabolism in Candida strains.

19F nuclear magnetic resonance was used for a noninvasive and quantitative study of flucytosine (FC; 5-fluorocytosine) metabolism in two strains of Candida albicans and one strain of Candida tropicalis with various susceptibilities to FC. Three intracellular fluorinated metabolites were detected in the highly susceptible strain, F-nucleotides (Fnt), F-nucleosides, and 5-fluorouracil (5FU). Fnt were partially converted into 5FU when the spectra of the yeasts were recorded at 37 degrees C without perfusion, but the intensities of the signals were not modified at 4 or 37 degrees C if the cells were perfused. In the acid extract, the Fnt signal was resolved into three distinct peaks; none of them was attributable to 5-fluoro-2'-deoxyuridine-5'-monophosphate. The same signals were detected in the partially resistant strain, but only 5FU was observed in the highly resistant strain; the resistance of the latter strain therefore was primarily due to a defect in UMP pyrophosphorylase. At the end of the incubation period, only FC and released 5FU were present in the culture media. The concentration of the intracellular fluorinated metabolites was increased if the strain was susceptible to FC. The total amount of metabolized FC was very similar for the highly susceptible and the partially resistant strains, but the percentage of Fnt was much higher in the former (38%) than in the latter (8%); the mild resistance of the partially resistant strain therefore was attributed to the decreased activity of UMP pyrophosphorylase.

Drug resistance in the opportunistic pathogens Candida albicans and Candida glabrata.

There are three major classes of antifungal drug used to treat patients suffering from topical and systemic infections caused by Candida albicans. Both the polyene macrolide antibiotics and the synthetic imidazole derivatives interact with membranes of sensitive organisms causing an impairment of function and cessation of growth. It is possible to obtain mutants of C. albicans resistant to these drugs but they are not a clinical problem. This may result from the fact that the organism is diploid with no haploid stage in its life cycle and the interaction of these compounds with their target is complex involving a number of membrane constituents. In contrast the occurrence of strains of C. albicans resistant to 5-fluorocytosine is a serious clinical problem. Here partial resistance is associated with heterozygosity at the locus coding for UMP pyrophosphorylase. Mitotic segregation can give rise to homozygous resistance in strains where the enzyme is completely absent. This is analogous to acyclovir resistance in herpes simplex virus where resistance is associated with loss of the virus encoded thymidine kinase.

Complementation analysis of resistance to 5-fluorocytosine in Candida albicans.

A complementation test was devised to study allelism among the genetic determinants of resistance to 5-fluorocytosine in Candida albicans. Complementation was demonstrated in control hybrids produced by crossing a resistant strain that was deficient in cytosine deaminase activity with four other resistant strains deficient in UMP pyrophosphorylase activity. This complementation test was used to test allelism of the resistance determinants present in five clinical isolates. All were found to bear recessive alleles of the locus (FCY1) that determined 5-fluorocytosine resistance associated with low levels of UMP pyrophosphorylase activity.

Decreased activity of UMP pyrophosphorylase associated with resistance to 5-fluorocytosine in Candida albicans.

UMP pyrophosphorylase activity was assayed in crude lysates prepared from Candida albicans. Specific activity of UMP pyrophosphorylase was high in clinical isolates which were susceptible to 5-fluorocytosine. Resistant clinical isolates displayed low activity, and partially resistant (heterozygous) isolates displayed intermediate activity. Segregation from the heterozygous state resulted in a homozygous susceptible segregant with high UMP pyrophosphorylase activity and a homozygous resistant segregant with low activity. The observed specific activities were consistent with the hypothesis that specific activity was determined by the sum of the activities due to the dominant (FCY) and recessive (fcy) alleles of the resistance gene. Strains which possessed little UMP pyrophosphorylase activity released uracil into the medium when grown in the absence of 5-fluorocytosine; this result suggested that recycling of intracellular uracil is a normal function of this enzyme.

UMP pyrophosphorylase of Tetrahymena pyriformis: Partial purification and properties

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 m m, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.

Uracil transport in Saccharomyces cerevisiae.

Uracil transport inSaccharomyces cerevisiae is mediated by a specific “permease” which does not recognize other pyrimidines such as uridine, cytosine, thymine, 2-hydroxypyrimidine or 5-amino-uracil; hypoxanthine and 6-amino-uracil slightly inhibit the uptake of uracil in a strain lacking cytosine permease activity. Wild type cells concentrate extracellular uracil before its transformation into UMP and subsequent incorporation into nucleic acids. A strain lacking UMP pyrophosphorylase and uridine ribohydrolase (strainfur 1–8 rh, in which the endogenous production as well as the utilization of uracil are lacking) is able to concentrate14C-2 uracil from the medium. At the same time no other14C-2 labelled compound could be detected in this strain, thus suggesting that the uptake of uracil in yeast occurs by active transport which is not coupled to the UMP pyrophosphorylase. The optimal pH of uracil uptake in standard growth conditions was 4.3. It was deduced from experiments performed on strainfur 1–8 rh with3H-5 and14C-2 uracil that the intracellular pool of uracil is recycled once the steady-state has been reached. First order kinetics with similar rate constants were observed for uracil efflux in strainfur 1–8 rh (k min−1=0.75±0.08) as well as in the strain lacking uracil permease,fur 1–8 rh fur 4–6 (k min−1=0.60±0.08). The intracellular pool of14C-2 uracil can be chased in strainfur 1–8 rh by addition of3H uracil without inducing a large initial acceleration of the exit rate (the rate constant remained at 0.60). 2-4-dinitrophenol inhibits the uptake of uracil but also reduces the efflux of uracil in strainfur 1–8 rh fur 4–6. These data and the comparison with cytosine transport in the same organism support the hypothesis that, whereas uracil uptake is a “permease” mediated active transport, the efflux of uracil does not involve the uracil uptake “permease”. A coefficient of permeability of 7.4×10−7 cm sec−1 was calculated for uracil.

Studies on the mechanism of resistance to 5-fluorocytosine, an antifungal agent, in fungi.

The mechanism of 5-fluorocytosine (5-FC) resistance was studied in highly resistant mutants isolated from Saccharomyces cerevisiae ATCC 9763. The development of resistance to 5-FC in these strains was caused by the obstruction of further enzymatic conversion of 5-fluorouracil (5-FU), produced by deamination of 5-FC in the cells, due to the lack of UMP pyrophosphorylase. Consequently, 5-FC was not incorporated into RNA, the most probable site of the antifungal activity of 5-FC. The 5-FU thus formed in the cells was excreted rapidly into the medium, thereby the resistant strains effectively remove toxic effects, if any, of the derived 5-FU from the cells.The mechanism for the simultaneous acquisition of the cross-resistance to 5-FU was also elucidated by the differences in the abilities to incorporate 5-FU by the sensitive and resistant strains.The similar mechanism of 5-FC resistance was also predicted in the case of Fonsecaea pedrosoi, a clinical isolate.

Inhibitory effects of 2-thiouracil and 2-thiouracil metabolites on enzymes involved in pyrimidine nucleotide biosynthesis

The inhibitory effects of thiouracil, thiouridine and thio-UMP on enzymes in the pyrimidine pathways leading to nucleic acid synthesis were studied. Thiouracil. but not thiouridine. was a potent competitive inhibitor of rat liver uridine phosphorylase. However, thiouridine was a potent competitive inhibitor of rat liver uridine kinase, whereas thiouracil and other antithyroid drugs produced no effect. Thio-UMP was a weak inhibitor of this enzyme but was less active than UMP. Thiouracil and thio-UMP produced little inhibition of bacterial UMP pyrophosphorylase, although thiouracil is a good substrate. Thiouracil and its analogs had no measurable effect on calf thymus or baker's yeast OMP pyrophosphorylase, but thio-UMP inhibition of rat liver OMP decarboxylase was comparable to that obtained with UMP. The results presented demonstrate that there are three sites in the pyrimidine pathways where thiouracil or its metabolites may exert substantial inhibition. The enzymes primarily affected are uridine phosphorylase and uridine kinase which are inhibited by thiouracil and thiouridine, respectively, and OMP decarboxylase which is inhibited by thio-UMP. These results support the hypothesis that some of the biological effects of thiouracil may be due to an alteration in pyrimidine nucleotide synthesis.

Relative antithyroid effects of 2-thiouracil, 2-thiouridine and 2-thio-UMP.

SummaryThe presence of uridine phosphorylase and thymidine phosphorylase, enzymes capable of converting thiouracil to thiouridine, was demonstrated in thyroid tissue from several species. Uridine kinase which catalyzes further metabolism of thiouridine to thio-UMP was also present. The activity of UMP pyrophosphorylase which converts thiouracil to thio-UMP in a one-step reaction was insignificant.Comparisons of antithyroidal activity in intact rats and with partially purified porcine thyroid peroxidase demonstrate that thiouridine was approximately 10% as potent as thiouracil in vivo but less than 1% as active as thiouracil as an inhibitor of thyroid peroxidase. Thio-UMP was about 5% as active as thiouracil on thyroid peroxidase.These results indicate that it is highly unlikely that thiouracil conversion to a nucleoside or nucleotide is involved in its antithyroidal action.

Conversion of the antithyroid drug 2-thiouracil to 2-thio-5′-UMP by UMP pyrophosphorylase

The metabolism of 2-thiouracil by UMP pyrophosphorylase was investigated. The enzyme utilized 2-thiouracil, but not methylthiouracil, propylthiouracil, or methimazole, as a substrate. The reaction product displayed chromatographic properties, uv-absorption characteristics and ratios of thiouracil, ribose, and phosphate identical to those of 2-thio-5′-UMP. In addition, the product was a substrate for 5′-nucleotidase forming 2-thiouridine. Based on this evidence the product was identified as 2-thio-5′-UMP. The conversion of thiouracil to thio-UMP was shown to be associated with UMP pyrophosphorylase activity rather than OMP pyrophosphorylase, to have a specific requirement for PRPP, to have an optimal pH of about 7.8 for thiouracil as opposed to an optimum of about 6.2 for uracil, and to be markedly depressed by high concentrations of both K and Na ions. Kinetic studies with the partially purified enzyme demonstrated a K m value for thiouracil of 6.7 × 10 −4 m compared to a value of 7.7 × 10 −6 m for uracil.

An unusual electrocardiographic pattern of myocardial infarction. (Type QT 1C 5–6) : Puddu, V., Mussafia, A., and Giordano, G.: Cardiologia 11: 133, 1947

The metabolism of 2-thiouracil by UMP pyrophosphorylase was investigated. The enzyme utilized 2-thiouracil, but not methylthiouracil, propylthiouracil, or methimazole, as a substrate. The reaction product displayed chromatographic properties, uv-absorption characteristics and ratios of thiouracil, ribose, and phosphate identical to those of 2-thio-5′-UMP. In addition, the product was a substrate for 5′-nucleotidase forming 2-thiouridine. Based on this evidence the product was identified as 2-thio-5′-UMP.The conversion of thiouracil to thio-UMP was shown to be associated with UMP pyrophosphorylase activity rather than OMP pyrophosphorylase, to have a specific requirement for PRPP, to have an optimal pH of about 7.8 for thiouracil as opposed to an optimum of about 6.2 for uracil, and to be markedly depressed by high concentrations of both K and Na ions. Kinetic studies with the partially purified enzyme demonstrated a Km value for thiouracil of 6.7 × 10−4m compared to a value of 7.7 × 10−6m for uracil.