Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 1: discovery of novel orally active inhibitors of human thymidine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been prepared as inhibitors of human thymidine phosphorylase (TP). To enhance the in vivo antitumor activity of fluorinated pyrimidine 2'-deoxyribonucleosides such as 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd), a potent TP inhibitor preventing their degradation to an inactive compound, has become a target of medicinal chemistry. We present here the synthesis and evaluation of novel human TP inhibitors. Introduction of an N-substituted aminomethyl side chain at the 6-position of 5-chlorouracil has improved water solubility and enhanced inhibitory activity compared with the known TP inhibitor, 6-amino-5-chlorouracil. Compound 42 was reasonably well absorbed in mice after oral administration. When combined with F(3)dThd, compound 42 exerted its TP inhibitory potency by increasing the maximum plasma concentrations of the former as evidenced in experiments with monkeys. Positive changes in pharmacokinetic profile were accompanied by the enhanced in vivo antitumor activity of this combination when compared to F(3)dThd alone, in mice bearing human tumor xenografts. Both biochemical and pharmacological effects appeared to fit the concept as anticipated.     

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.     

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.     

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.     

Activity and substrate specificity of pyrimidine phosphorylases and their role in fluoropyrimidine sensitivity in colon cancer cell lines

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5'-deoxy-5-fluorouridine (5'DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect. The high TP expressing Colo320TP1 cells were most sensitive to 5'DFUR and 5FU, with IC50 values of 1.4 and 0.2 microM, respectively, while SW948 and SW1398 were insensitive to 5'DFUR (IC50>150 microM for 5'DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC(50) for 5'DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5'DFUR; p<0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5'DFUR and 5FU separately. HaCaT might be a model for 5'DFUR toxicity. In the colon cancer cells 5'DFUR degradation varied from 0.4 to 50 nmol 5FU/h/10(6)cells, that of TdR from 0.3 to 103 nmol thymine/h/10(6)cells, that of Urd from 0.8 to 79 nmol uracil/h/10(6)cells, while conversion of 5FU to FUrd was from 0.3 to 46 nmol/h/10(6)cells. SW948 and SW1398 were about equally sensitive to 5'DFUR and 5FU, but SW1398 had higher phosphorylase activity (>65-fold) compared to SW948. In SW948 and HaCaT TPI and BAU inhibited TdR and Urd phosphorolysis (>80%), respectively. Both TP and UP contributed to the phosphorolysis of 5'DFUR and 5FU. In the presence of both inhibitors, still phosphorolysis of 5FU (>40%) was detected in the tumor and HaCaT cell lines, and remarkably, that of all four substrates in SW1398 cells. 5'DFUR phosphorolysis was also measured in situ, where Colo320TP1, SW1398, and HaCaT cells produced significant amounts 5FU from 5'DFUR (>10 nmol/24h/10(6)cells). In Colo320TP1 and in HaCaT cells TPI completely prevented 5FU production, but not in SW1398 cells, where BAU decreased this by 67% (p<0.01). High uracil and dUrd levels were detected in the medium. Uracil accumulation was heavily reduced in the presence of TPI for Colo320TP1 and HaCaT cells, whereas 5FU-induced dUrd production by these cell lines increased (p<0.01). In contrast, for SW1398 cells only BAU was able to reduce uracil levels, and dUrd production remained unchanged. In conclusion, overlapping substrate specificity was found for TP and UP in the cell lines, in which both enzymes were responsible for converting TdR and Urd, and 5'DFUR. 5'DFUR and 5FU were converted to their products in both the colon cancer cells and keratinocytes.     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma

We investigated the biological role of thymidine phosphorylase (TP), an angiogenic factor, in gastric cancer cell migration and invasion and explored a therapeutic approach for high TP-expressing tumors using TP enzymatic inhibitor (TPI) and rapamycin. We established TP cDNA overexpressing gastric cancer cell lines (MKN-45/TP and YCC-3/TP) and did invasion and adhesion assays with Matrigel-coated transwell membranes. The related signal pathway using recombinant human TP (rhTP), deoxy-d-ribose (D-dRib), and signal pathway inhibitors (wortmannin, LY294002, and rapamycin) was investigated. First, AGS and MKN-1 gastric cancer cell lines showed dose-dependent up-regulation of invasiveness through Matrigel following treatment with rhTP or D-dRib. TP-overexpressing cancer cell lines displayed increased migration and invasion activity, which doubled with rhTP and D-dRib treatment. This activity depended on the enzymatic activity of TP, and TP stimulated the adhesion of cancer cells onto Matrigel and induced actin filament remodeling. Finally, we showed that this activity is related to increased phosphatidylinositol 3-kinase activity in TP-overexpressing cells and that combination treatment with rapamycin and TP enzymatic inhibitor produces an additive effect to abrogate TP-induced invasion. Taken together, TP increases the migration and invasion of gastric cancer cells, especially in TP-expressing cells. Therapies targeting TP might diminish the propensity for invasion and metastasis in gastric cancer.     

The role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in tumor behavior

Platelet-derived endothelial cell growth-factor (PD-ECGF) is similar to the pyrimidine enzyme thymidine phosphorylase (TP). A high TP expression at tumor sites is correlated with tumor growth, induction of angiogenesis, and metastasis. Therefore, high TP is most likely associated with a poor prognosis. TP is not only expressed in tumor cells but also in tumor surrounding tissues, such as tumor infiltrating macrophages. TP catalyzes the conversion of thymidine to thymine and doxyribose-1-phosphate (dR-1-P). The latter in its parent form or in its sugar form, deoxyribose (dR) may play a role in the induction of angiogenesis. It may modulate cellular energy metabolism or be a substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose (L-dR) and thymidine phosphorylase inhibitor (TPI) can reverse these effects. The mechanism of TP induction is not yet completely clear, but TNF, IL10 and other cytokines have been clearly shown to induce its expression. The various complex interactions of TP give it an essential role in cellular functioning and, hence, it is an ideal target in cancer therapy.     

Inhibitory potency of 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one nucleosides against uridine phosphorylase from mouse leukemic L1210 cells

The inhibitory activity of a series of novel sugar-modified nucleosides derived from 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one against uridine phosphorylase purified from mouse leukemic L-1210 cells was investigated. Significant activity was encountered with O2,2'-anhydro-5-benzylcytidine hydrochloride, 2',3'-dideoxy-5-benzyluridine, 2',3'-dideoxy-4-thiouridine and alpha- and beta-anomers of 5-benzyl-1-(2-deoxy-D-arabino-hexopyranosyl)uracil.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Using of 4-thiouridine and 4-thiothymidine for pyrimidine nucleoside phosphorylase studing

4-Thiouridine and 4-thiothymidine were developed as efficient substrates for spectrophotometric determination of uridine phosphorylase and thymidine phosphorylase activity. 4-Thiouridine has maximum absorbance at 330 nm (pH 7.5). The change in extinction coefficient for 4-thiouridine/4-thiouracil, deltaepsilon is 3000 M(-1) x cm(-1). It appeared that 4-thiouridine is a good substrate for uridine phosphorylase with Michaelis-Menten constant 130 microM and kcat 49 s(-1). In the case of 4-thiothymidine/4-thiothymine deltaepsilon is even larger: 5000 M(-1) x cm(-1) at 336 nm.     

Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.     

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.