Crystal structures of a new class of pyrimidine/purine nucleoside phosphorylase revealed a Cupin fold.

Nucleotides metabolism is a fundamental process in all organisms. Two families of nucleoside phosphorylases (NP) that catalyze the phosphorolytic cleavage of the glycosidic bond in nucleosides have been found, including the trimeric or hexameric NP-I and dimeric NP-II family enzymes. Recent studies revealed another class of NP protein in Escherichia coli named Pyrimidine/purine nucleoside phosphorylase (ppnP), which can catalyze the phosphorolysis of diverse nucleosides and yield d-ribose 1-phosphate and the respective free bases. Here, we solved the crystal structures of ppnP from E. coli and the other three species. Our studies revealed that the structure of ppnP belongs to the RlmC-like Cupin fold and showed as a rigid dimeric conformation. Detail analysis revealed a potential nucleoside binding pocket full of hydrophobic residues, and the residues involved in the dimer and pocket formation are all well conserved in bacteria. Since the Cupin fold is a large superfamily in the biosynthesis of natural products, our studies provide the structural basis for understanding, and the directed evolution of NP proteins.

pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 106 for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.

Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007.

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Enzymes of pyrimidine salvage pathways in intraerythrocytic Plasmodium falciparum.

Malaria remains a significant public health problem worldwide with an estimated annual global incidence of 200 million and an estimated 450,000 annual deaths. Among the five known human malarial species, Plasmodium falciparum is the deadliest and most resistant to antimalarials. Hence, there is a need for new antimalarial targets. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and their hosts. In view of their high rate of replication, parasites require very active nucleic acid synthesis which necessitates large supplies of the indispensable pyrimidine nucleotides. Consequently, delineation of P. falciparum pyrimidine metabolic pathways may reveal potential targets for the chemotherapy of malaria. Previous studies reported the existence of pyrimidine de novo pathways in this organism. The present results demonstrate the presence of enzymes of the pyrimidine salvage pathways in P. falciparum and indicate that this parasite is capable of pyrimidine salvage. Furthermore, some of the pyrimidine salvage enzymes, e.g., dTMP kinase, phosphoribosyltransferase, and uridine phosphorylase could be excellent targets for chemotherapeutic intervention against this parasite.

Crystal structure of pyrimidine-nucleoside phosphorylase from Bacillus subtilis in complex with imidazole and sulfate.

Pyrimidine-nucleoside phosphorylase catalyzes the phosphorolytic cleavage of thymidine and uridine with equal activity. Investigation of this protein is essential for anticancer drug design. Here, the structure of this protein from Bacillus subtilis in complex with imidazole and sulfate is reported at 1.9 Šresolution, which is an improvement on the previously reported structure at 2.6 Šresolution. The localization and position of imidazole in the nucleoside-binding site reflects the possible binding of ligands that possess an imidazole ring.

A thermostable pyrimidine nucleoside phosphorylase from Brevibacillus borstelensis LK01 for synthesizing halogenated nucleosides.

OBJECTIVE: To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining. RESULTS: BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2'-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61-84%. CONCLUSION: An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

[Purine and pyrimidine nucleoside phosphorylases - remarkable enzymes still not fully understood]. / Nukleozydowe fosforylazy purynowe i pirymidynowe - niezwykle enzymy ciagle nie do konca rozszyfrowane.

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

The pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis and how it may affect nucleoside-based therapy.

Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Inhibition of pyrimidine and purine nucleoside phosphorylases by a 3,5-dichlorobenzoyl-substituted 2-deoxy-D-ribose-1-phosphate derivative.

The 3,5-dichlorobenzoyl-substituted 2-deoxy-D-ribose-1-phosphate derivative, designated Cf2891, was found to inhibit a variety of pyrimidine and purine nucleoside phosphorylases (NPs) with preference for uridine- and inosine-hydrolyzing enzymes [uridine phosphorylase (UP; EC 2.4.2.3), pyrimidine nucleoside phosphorylase (PyNP; EC 2.4.2.2) and purine nucleoside phosphorylase (PNP; EC 2.4.2.1)]. Kinetic analyses revealed that Cf2891 competes with inorganic phosphate (P(i)) for binding to the NPs and, depending on the nature of the enzyme, acts as a competitive or non-competitive inhibitor with regard to the nucleoside binding site. Also, the compound prevents breakdown of pyrimidine analogues used in the treatment of viral infections and cancer. Since NPs are abundantly present in tumor tissue and may be overexpressed due to secondary bacterial infections in immunocompromised patients suffering viral infections, Cf2891 may serve as a lead molecule for the development of inhibitors to be used in nucleoside-based combination therapy.

Phosphoramidate ProTides of the anticancer agent FUDR successfully deliver the preformed bioactive monophosphate in cells and confer advantage over the parent nucleoside.

The fluorinated pyrimidine family of nucleosides continues to represent major current chemotherapeutic agents for treating solid tumors. We herein report their phosphate prodrugs, ProTides, as promising new derivatives, which partially bypass the dependence of the current drugs on active transport and nucleoside kinase-mediated activation. They are also resistant to metabolic deactivation by phosphorolytic enzymes. We report 39 ProTides of the fluorinated pyrimidine FUDR with variation in the aryl, ester, and amino acid regions. Notably, only certain ProTide motifs are successful in delivering the nucleoside monophosphate into intact cells. We also find that the ProTides retain activity in mycoplasma infected cells, unlike FUDR. Data suggest these compounds to be worthy of further progression.

Pyrimidine salvage pathway in Mycobacterium tuberculosis.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.

The relationship between clinicopathological factors and the reduction of pyrimidine nucleoside phosphorylase activity after preoperative administration of 5'-deoxy-5-fluorouridine.

AIM: The response to fluoropyrimidine chemotherapeutic drugs is different in individual tumors. Predictive biomarkers of antitumor effects by these drugs are unknown. 5'-Deoxy-5-fluorouridine (5'-DFUR), a fluoro-pyrimidine chemotherapeutic drug, is converted to 5-fluorouracil (5-FU) by pyrimidine nucleoside phosphorylase (PyNPase). It is suggested that 5'-DFUR will efficiently exert antitumor effects via PyNPase in tumor tissues. The change of PyNPase activity in tumor tissues following 5'-DFUR administration may reflect antitumor effects, and may be useful for detecting predictive factors of antitumor effects. The aim of this study was to search for predictive factors of antitumor effects by analyzing the relationship between clinicopathological factors and the change of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration. PATIENTS AND METHODS: PyNPase activity in colorectal tissues from 45 patients with colorectal tumors was measured using an ELISA method. RESULTS: The reduction rate of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration was correlated with significant differences in lymphatic invasion, stage, and histologic classification. It is suggested that lymphatic invasion, stage (distant metastasis), and histologic classification may be predictive factors for evaluating antitumor effects and selecting 5-FU-based chemotherapeutic drugs for patients with colorectal tumors.

[Significance of pyrimidine nucleoside phosphorylase and dihydropyrimidine dehydrogenase levels to predict postoperative recurrence in primary breast cancer].

BACKGROUND: High expression of PyNPase (pyrimidine nucleoside phosphorylase) and DPD (dihydropyrimidine dehydrogenase) in breast cancer has been reported. Breast cancer patients with high expression of PyNPase reportedly have a poor prognosis. METHODS: We evaluated the relationship between postoperative prognosis, and clinicopathological factors including HER2 expression and the levels of PyNPase and DPD in breast cancer. PyNPase and DPD levels in tumors and nontumorous tissues were examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: PyNPase and DPD levels in tumors were significantly higher than in non-tumorous tissues (p<0.001). The DPD levels in tumors associated with> or =2+expression of HER2 were significantly higher than in others (p=0.014). Disease-free survival in patients with<100 U/mg protein of PyNPase levels or<4 of PyNPase/DPD ratio was significantly better compared with others (p=0.022, p=0.014). CONCLUSION: PyNPase/DPD ratio may be a new prognostic marker in breast cancer.

Tissue levels of pyrimidine nucleoside phosphorylase activity in human and rodent bladder cancer and normal bladder tissue.

OBJECTIVES: To assess the relationship between the tissue levels of pyrimidine nucleoside phosphorylase (PyNpase) and clinicopathological parameters in human bladder cancer and to investigate the PyNpase levels in rat and mouse urinary bladder initiated by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). METHODS: The PyNpase levels in tumor tissue, normal tissue adjacent to the tumor, and normal tissue apart from the tumor were measured in 102 patients. Additionally, the PyNpase levels were measured in rat and mouse urinary bladders treated with BBN. RESULT: The PyNpase levels of tumor tissue significantly correlated to the tumor grade and growth pattern (papillary/non-papillary), while stage, multiplicity, and tumor shape (peduncle/sessile) were not independent factors. The low-risk tumor of primary, single, G1-Ta showed significantly low levels of PyNpase. The PyNpase levels in the tumor tissue were significantly higher than those in the normal tissue. The PyNpase levels in the adjacent normal tissue were significantly higher than those in the distant normal tissue. The PyNpase levels in rat bladder tissue were significantly higher in the BBN-treatment groups than in those in the control group, only during the early carcinogenic stage. The PyNpase levels in mouse bladder tissue were significantly higher in BBN-treatment groups than in those in the control group during the whole experiment period. CONCLUSION: Our results indicated that not only tumor tissue but also normal tissue adjacent to the tumor had a potential of angiogenesis for tumor development, and transurethral resection of the bladder tumor with a wide normal margin seems to be a reasonable strategy for decreasing the risk of recurrence.

Pyrimidine nucleoside phosphorylase and dihydropyrimidine dihydrogenase activities as predictive factors for the efficacy of doxifluridine together with mitomycin C as adjuvant chemotherapy in primary colorectal cancer.

BACKGROUND/AIMS: Pyrimidine Nucleoside Phosphorylase (PyNPase) converts 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine) to 5-fluorouracil (5-FU). While this reaction is taking place Dihydropyrimidine Dihydrogenase (DPD) catalyzes 5-FU to inactive molecules. Mitomycin C (MMC) elevates the PyNPase level in tumor cells. METHODOLOGY: We investigated 17 colorectal cancer patients' PyNPase and DPD activities in tumor and normal tissues using an enzyme-linked immunosorbent assay (ELISA) to assess their clinical significance as indicators for selecting colorectal cancer patients for 5'-DFUR together with MMC as adjuvant chemotherapy. RESULTS: Six of 17 patients developed experienced a recurrence. Tumor DPD activity of the 6 patients who had a recurrence were higher than those of the 11 patients with no recurrence (p = 0.047). On the other hand, there were no significant differences in both the PyNPase and the PyNPase/DPD (P/D) ratio between the group with recurrence and the group without recurrence. For survival analyses, we designed the cut-off value of tumor PyNPase, DPD and P/D ratio as their median value and classified patients into a higher group and a lower group, but there were no significant differences between the groups. CONCLUSIONS: The DPD activity in the tumor may be a useful indicator for selecting patients likely respond to 5'-DFUR together with MMC as adjuvant chemotherapy. If tumor DPD is high, we had better select a different anticancer drug.

Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8.

Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure-thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291 K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8 A resolution using synchrotron radiation. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 58.83, b = 76.23, c = 103.86 A, beta = 91.3 degrees.

[Clinical significance of pyrimidine nucleoside phosphorylase (PyNPase) in breast cancer].

To clarify the clinical significance of pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer, we examined the possible correlation of PyNPase activity to clinicopathological features and prognosis in twenty-one patients with primary breast cancer from April 2000 to December 2001. Flow signals of tumors were analyzed by Power Doppler sonography (PDUS), and maximal velocity (V(max)) was calculated. PyNPase activity of resected specimens was assayed by ELISA method. PyNPase activities in resected cancerous tissue were 156.9+/-63.5 unit/mg (mean+/-SD), which were significantly higher than that in normal tissue (19.0+/-18.1 unit/mg, p<0.0001). PyNPase activity was positively correlated with tumor size (r=0.496, p=0.026) and V(max) (r=0.498, p=0.021). The disease free survival rate was significantly lower in the high PyNPase activity group than in the low PyNPase activity group. In overall survival rate, there was no significant difference between the high and low PyNPase activity groups. In the multivariate analysis, PyNPase activity was an independent predictor of postoperative recurrence (p=0.032). We suggest that PyNPase activity is associated with progression and proliferation of breast cancer, and that it may be useful for prediction of the prognosis.