Studies on a pyrimidine phosphoribosyltransferase from murine leukemia P1534J. Partial purification, substrate specificity, and evidence for its existence as a bifunctional complex with orotidine 5-phosphate decarboxylase.

A pyrimidine phosphoribosyltransferase, previously shown to utilize 5-fluorouracil and possibly also uracil and orotate (Reyes, P. (1969) Biochemistry 8, 2057-2062), has been purified about 100-fold from murine leukemia P1534J. Roughly 20% of the original activity was recovered to yield an enzyme preparation with a specific activity of 7.4 mumol of 5-fluorouracil utilized/hour/mg of protein. Disc gel electrophoresis of this preparation revealed the presence of a major band of protein accompanied by several trace contaminants. Emphasis was placed on a study of the substrate specificity of this enzyme. 5-Fluorouracil, uracil, and orotate phosphoribosyltransferase activities purified in parallel during fractionation with ammonium sulfate and protamine sulfate and eluted together from columns of Sephadex tG-150 and DEAE-cellulose. The three phosphoribosyltransferase activities eluted from the Sephadex columns with an apparent molecular weight of 55,000 to 60,000. In spite of this coordinate fractionation, preferential losses of orotate activity were experienced during DEAE-cellulose chromatography. Orotate activity continued to behave in a unique manner under other conditions, such as during proteolytic digestion. In the latter case, however, all three activities responded in parallel when digestion took place in the presence of 5mM UMP. The following results provided additional evidence to support the view that all three phosphoribosyltransferase activities may be catalyzed by the same enzyme: (a) the apparent Km for 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) did not change significantly when enzyme activity was measured with either 5-fluorouracil, uracil, or orotate; (b) 5-fluorouracil and uracil were found to be mutually competitive inhibitors; the effect of 5-fluorouracil on orotate activity was likewise competitive in nature; (c) in the absence of UMP, orotate was a noncompetitive inhibitor of 5-fluorouracil and uracil activities, but in the presence of 5mM UMP it became a competitive inhibitor of both of these activities; (d) 5-fluorouracil and orotate activities co-sedimented in 5 to 20% sucrose gradients (uracil activity was not examined); and (e) a wide variety of normal mouse tissues displayed virtually the same 5-fluorouracil to uracil to orotate activity ratio as found in P1534J enzyme preparations. The apparent Km and Ki values reported in this study indicate that the preferred pyrimidine substrate is orotate. It seems likely, therefore, that this enzyme functions in vivo as an orotate phosphoribosyltransferase. Orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities (a) eluted together during gel filtration on Sephadex G-150, (b) co-sedimented in 5 to 20% sucrose gradients, (c) remained associated during fractionation with ammonium sulfate and protamine sulfate, and (d) separated into a phosphoribosyltransferase and decarboxylase component when enzyme preparations previously subjected to limited proteolysis by elastase were sedimented in sucrose gradients...     

Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus.

We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.     

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl₂ for maximum activity but was inhibited by MnCl₂; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K_m values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 μm and 11 μm; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5′-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5′-phosphate and 5-fluorouridine 5′-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.     

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells

The de novo biosynthesis of pyrimidine nucleotides is completed by two sequential enzyme activities that convert orotate plus 5-phosphoribosyl-1-pyrophosphate to orotidine-5′-monophosphate (OMP) and PP_i and then decarboxylate OMP to produce 5′-uridylic acid. In mammalian cells the two enzyme activities, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, form a normally inseparable enzyme complex. It was previously reported that this complex is able to channel the intermediate product, OMP (Traut, T. W., and Jones, M. E., 1977, J. Biol. Chem.252, 8374–8381). The studies reported here indicate that one advantage of this channeling of OMP is to spare OMP from being degraded to orotidine by a potentially competitive nucleotidase activity. Yeast cells have two separate enzymes instead of an enzyme complex, and lack the ability to channel OMP. The OMP formed in yeast cells is not degraded because these cells lack significant nucleotidase activity. These results suggest that the capability for channeling OMP may have been important in evolving the enzyme complex found in mammalian cells.     

A reexamination of the substrate utilization of 2-thioorotidine-5'-monophosphate by yeast orotidine-5'-monophosphate decarboxylase

A potential alternate substrate for orotidine-5'-monophosphate decarboxylase, 2- thio-orotidine-5'-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, (31)P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10(-7) from the k(cat) for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 microM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.     

Enzymatic synthesis of [6-14C]orotidine 5'-monophosphate and its use in the assay of orotate phosphoribosyltransferase and orotidylate decarboxylase

A rapid and efficient method is described for the synthesis of [6-¹⁴C]orotidine 5′-monophosphate from radioactive orotic acid using purified yeast orotate phosphoribosyltransferase and inorganic pyrophosphatase. Radioactive orotidine 5′-monophosphate is purified by ion exchange chromatography and employed in small scale assays of Drosophila orotate phosphoribosyltransferase and orotldylate decarboxylase in which both enzyme activities are simultaneously measured in single reaction mixtures. Radioactive substrate and products are separated for counting using DEAE-cellulose paper chromatograms developed in one or two solvents.     

UMP synthesis in the kinetoplastida

All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular form of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmi, whereas orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.     

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.     

The derepression of enzymes of de novo pyrimidine biosynthesis pathway in Brevibacterium ammoniagenes producing uridine-5-monophosphate and uracil

A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyltransferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.     

6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity

6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine-5′-monophosphate and 6-azauridine-5′-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6-azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and ³¹P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.     

Potential for ambiguity in the interpretation of immunochemical studies on multienzymic proteins. The case of uridylate synthase

Rabbit antibodies directed against homogeneous uridylate synthase multienzyme from mouse Ehrlich ascites carcinoma precipitate both the orotidine-5'-monophosphate decarboxylase (EC 4.1.1.23) and orotate phosphoribosyltransferase (EC 2.4.2.10) activities of mouse and human erythrocyte uridylate synthase. When the partially purified human enzyme is used as antigen the two activities coprecipitate with the same apparent titer; however, when the mouse carcinoma protein was studied under the same conditions the decarboxylase activity immunoprecipitated with significantly higher avidity than did the transferase activity. Since the mouse multienzyme has been shown to be a single polypeptide that contains both activities (McClard, R.W., Black, M.J., Livingstone, L.R. and Jones, M.E. (1980) Biochemistry 19, 4699-4706), these results were, at face value, surprising. However, when the mouse orotate phosphoribosyltransferase activity (which is largely lost upon dilution into the immunoassay medium) was stabilized with 5-phosphoribosyl 1-pyrophosphate, both enzyme activities displayed the same apparent antibody titer. The immunochemical studies indicate that the antibodies, as a population, preferentially bind to a form or forms of the enzyme which contain(s) denatured transferase domains. A calculation based on a simple model yields a value of approximately 100 for the relative selectivity of the antibody for the denatured form of uridylate synthase. These results illustrate an ambiguity that is inherent in the interpretation of immunochemical studies on such multienzymic proteins; that is, it is possible to conclude incorrectly that two enzyme activities are not functionally associated if one of the catalytic domains is particularly unstable and thereby displays greater immunoreactivity for the specific antiserum.     

Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: a new bidirectional selective system for mammalian cells

We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible--in one experiment--to screen more than 10 of these cells for growth under selective media containing 10(-4) M 5-fluoroorotic acid and 10(-4) M uridine. Cells capable of sustained growth in such media were eventually recovered. Clones of these cells, unlike clones of the parental population, required uridine for growth and contained only 0.39% as much orotate phosphoribosyltransferase, less than 0.5% as much 5-fluoroorotate phosphoribosyltransferase, and 0.013% as much orotidine-5'-monophosphate decarboxylase activity as the parental clones. The parental and variant clones had similar levels of activity for six other enzymes that participate in pyrimidine metabolism.     

Direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate decarboxylase

New sensitive and direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate-5'-monophosphate (OMP) decarboxylase are described. The assays utilize a thioketone derivative of orotate (4-thio-6-carboxyuracil) which is converted into 4-thio-OMP by the transferase in the presence of phosphoribosyl pyrophosphate. 4-Thio-OMP is subsequently decarboxylated to 4-thio-UMP by OMP decarboxylase. A novel, efficient synthesis of thioorotate is described. Unlike the natural substrates, the interconversion of the thioketone derivatives yields large spectral changes in the near-visible absorption region. Orotate phosphoribosyltransferase is assayed at 333 nm with a molar extinction coefficient of 10,300 M-1 cm-1 for the conversion of thioorotate to either 4-thio-OMP or 4-thio-UMP. Orotidylate decarboxylase is assayed at 365 nm with a molar extinction coefficient of 3350 M-1 cm-1 for the conversion of 4-thio-OMP to 4-thio-UMP. Another advantage of these substrates is that they bind less tightly to orotate phosphoribosyltransferase and OMP decarboxylase than orotate or OMP, respectively. Thus, the initial rates of substrate conversion to product are readily measurable near the Km values for the thioketone substrates. The ability to follow the reactions directly permits the rapid determination of Km values for the thioketone substrates and Ki values for inhibitors of the enzymes.     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

Metabolism of pyrimidine bases and nucleosides in Neisseria meningitidis.

In Neisseria meningitidis, uridine, deoxyuridine, cytosine, cytidine, or deoxycytidine could not be used by uracil-requiring mutants as pyrimidine sources. Consistent with these findings, only 5-fluorouracil of the different fluoropyrimidine bases and nucleosides showed any inhibitory effect on the growth of four prototrophic strains of N. meningitidis. Likewise, only radioactive uracil was readily incorporated into nucleic acids, whereas uptake of radioactive uridine, cytosine, or cytidine could not be demonstrated. Uracil was converted to uridine 5'-monophosphate by uracil phosphoribosyltransferase, whereas enzyme activities for conversion of cytosine or any of the nucleosides were not detectable in meningococcal extracts.     

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase

The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.     

Isolation of uracil auxotrophs from Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Two uracil auxotrophs of the phytopathogen Burkholderia cepacia ATCC 25416, which is known to be involved in food spoilage, were isolated by a combination of ethylmethane sulphonate and D-cycloserine counterselection. One mutant exhibited depressed orotate phosphoribosyltransferase activity while the other mutant lacked orotidine 5'-monophosphate decarboxylase activity. Pyrimidine limitation of either auxotroph elevated aspartate transcarbamoylase and dihydroorotase activities by at least 1.5-fold indicating that these pathway enzymes may be repressible by a uracil-related compound in B. cepacia . Overall, regulation of de novo pyrimidine synthesis in the uracil auxotrophs of B. cepacia ATCC 25416 was observed.