Uridine Phosphorylase Research Articles

Proteomic Analysis of Renal Biomarkers of Kidney Allograft Fibrosis-A Study in Renal Transplant Patients.

Renal transplantation is the preferred treatment of end stage renal disease, but allograft survival is limited by the development of interstitial fibrosis and tubular atrophy in response to various stimuli. Much effort has been put into identifying new protein markers of fibrosis to support the diagnosis. In the present work, we performed an in-depth quantitative proteomics analysis of allograft biopsies from 31 prevalent renal transplant patients and correlated the quantified proteins with the volume fraction of fibrosis as determined by a morphometric method. Linear regression analysis identified four proteins that were highly associated with the degree of interstitial fibrosis, namely Coagulation Factor XIII A chain (estimate 18.7, adjusted p < 0.03), Uridine Phosphorylase 1 (estimate 19.4, adjusted p < 0.001), Actin-related protein 2/3 subunit 2 (estimate 34.2, adjusted p < 0.05) and Cytochrome C Oxidase Assembly Factor 6 homolog (estimate −44.9, adjusted p < 0.002), even after multiple testing. Proteins that were negatively associated with fibrosis (p < 0.005) were primarily related to normal metabolic processes and respiration, whereas proteins that were positively associated with fibrosis (p < 0.005) were involved in catabolic processes, cytoskeleton organization and the immune response. The identified proteins may be candidates for further validation with regards to renal fibrosis. The results support the notion that cytoskeleton organization and immune responses are prevalent processes in renal allograft fibrosis.

Immobilized enzyme reactors based on nucleoside phosphorylases and 2′-deoxyribosyltransferase for the in-flow synthesis of pharmaceutically relevant nucleoside analogues

In this work, a mono- and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the in-flow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2′-deoxy, 2′,3′-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine in 65–59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield).Both IMERs proved to be promising alternatives to chemical routes for the synthesis of nucleoside analogues. The developed in-flow system represents a powerful tool for the fast production on analytical scale of nucleosides for preliminary biological tests.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl–agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

X-Ray Structure and Molecular Dynamics Study of Uridine Phosphorylase from Vibrio cholerae in Complex with 2,2'-Anhydrouridine

The high-resolution three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complex with the competitive inhibitor 2,2'-anhydrouridine was determined by X-ray diffraction (RCSBPDB ID: 6RCA). The three-dimensional structure of this complex is compared with the previously determined structures of V. cholerae uridine phosphorylase in complex with the substrate (uridine) and S. typhimurium uridine phosphorylase in complex with 2,2'-anhydrouridine. The protein–inhibitor and protein–substrate binding free energies were calculated by the free-energy perturbation method. The number of stable hydrogen bonds between the 2,2'-anhydrouridine molecule and the active site of the enzyme is smaller and these bonds are longer compared to the natural substrate of the enzyme (uridine). However, calculations taking into account solvation energy of the molecule and the entropy effects showed that the binding of the inhibitor (2,2'-anhydrouridine) at the active site of the protein is energetically more favorable than the binding of the native substrate (uridine). These results may be useful in the design of new inhibitors with a higher selectivity for the binding sites of uridine phosphorylases.

A six‑gene support vector machine classifier contributes to the diagnosis of pediatric septic shock

Septic shock is induced by an uncontrolled inflammatory immune response to pathogens and the survival rate of patients with pediatric septic shock (PSS) is particularly low, with a mortality rate of 25–50%. The present study explored the mechanisms of PSS using four microarray datasets (GSE26378, GSE26440, GSE13904 and GSE4607) that were obtained from the Gene Expression Omnibus database. Based on the MetaDE package, the consistently differentially expressed genes (DEGs) in the four datasets were screened. Using the WGCNA package, the disease-associated modules and genes were identified. Subsequently, the optimal feature genes were further selected using the caret package. Finally, a support vector machine (SVM) classifier based on the optimal feature genes was built using the e1071 package. Initially, there were 2,699 consistent DEGs across the four datasets. From the 10 significantly stable modules across the datasets, four stable modules (including the magenta, purple, turquoise and yellow modules), in which the consistent DEGs were significantly enriched (P<0.05), were further screened. Subsequently, six optimal feature genes (including cysteine rich transmembrane module containing 1, S100 calcium binding protein A9, solute carrier family 2 member 14, stomatin, uridine phosphorylase 1 and utrophin) were selected from the genes in the four stable modules. Additionally, an effective SVM classifier was constructed based on the six optimal genes. The SVM classifier based on the six optimal genes has the potential to be applied for PSS diagnosis. This may improve the accuracy of early PSS diagnosis and suggest possible molecular targets for interventions.

A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP.

Bioinformatics of uridine/deoxyuridine paths in Trypanosoma evansi revealed targeting uridine phosphorylase and cytidine deaminase

The differences between the host and parasite metabolic paths could be a hot spot for discovery of antiparasitic targets. In this work, the metabolic paths of uridine and deoxyuridine in camels and the blood protozoan Trypanosoma evansi (T. evansi) were investigated by bioinformatics tools. While a set of de novo and salvage enzymes of uridine were found in camels, T. evansi was lacking uridine kinase and the source of UMP comes through salvage of uridine conversion to uracil. Therefore, inhibition of uridine phosphorylase (UPase) or cytidine deaminase (CDa) will affect the downstream UMP, dUMP and thymidylate synthesis by lowering the levels of uracil and uridine inside the parasite, respectively. Given the presence of two UPases in camel, low sequence similarity between camel and T. evansi UPases and CDase, targeting these enzymes might be without deleterious effects on the host cells.

Uridine phosphorylase 1 associates to biological and clinical significance in thyroid carcinoma cell lines.

Thyroid cancer incidence has been continuity increasing worldwide. Uridine phosphorylase 1 (UPP1) is a protein‐coding gene and has been detected that UPP1 was the higher expression in many solid malignancies, just as head and neck cancers, breast cancer, compared with paired normal tissue. But the act of UPP1 in thyroid cancer is not explicit. In this article, we investigate the function of UPP1 expression in thyroid cancer. The Cancer Genome Atlas (TCGA) unpaired thyroid cancer and normal RNA‐seq data were downloaded, and our paired thyroid cancer and normal samples were analysed by a polymerase chain reaction. The expression of UPP1 was regulated by transfected small interfering RNA, and the function of UPP1 was determined via migration, invasion and cell proliferation assays. Western blot assay was achieved to determine the UPP1 expression correlates with the function of 5‐FU regulate epithelial‐mesenchymal transition. The significant upregulation of UPP1 in thyroid cancer tissues compared with normal thyroid tissues was revealed by our data and TCGA data. UPP1 overexpression was significantly correlated with lymph node metastasis, tumour stage and tumour size. In the cell, experiments showed that UPP1 low expression significantly suppressed the migration, invasion and proliferation. Western blot assay proves the effect of UPP1 expression on 5‐FU regulates epithelial‐mesenchymal transition pathway. UPP1 plays a crucial oncogene in thyroid cancer. Our findings indicate that UPP1 might be a biomarker of thyroid cancer and may act by regulating epithelial‐mesenchymal transition (EMT).

Identification of an antilymphocyte transformation substance from Pasteurella multocida.

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai‐1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS‐PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT‐2 cells, which are an IL‐2‐dependent T cell line, nor did it modify IL‐2 production by Con A‐stimulated mouse spleen cells. The N‐terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai‐1 was cloned into expression vector pQE‐60 in Escherichia coli XL‐1 Blue. Recombinant UPase (rUPase) tagged with His at the C‐terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A‐stimulated mouse spleen cells and may be a virulence factor.

Spatially resolved metabolomics to discover tumor-associated metabolic alterations

Characterization of tumor metabolism with spatial information contributes to our understanding of complex cancer metabolic reprogramming, facilitating the discovery of potential metabolic vulnerabilities that might be targeted for tumor therapy. However, given the metabolic variability and flexibility of tumors, it is still challenging to characterize global metabolic alterations in heterogeneous cancer. Here, we propose a spatially resolved metabolomics approach to discover tumor-associated metabolites and metabolic enzymes directly in their native state. A variety of metabolites localized in different metabolic pathways were mapped by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In combination with in situ metabolomics analysis, this method provided clues into tumor-associated metabolic pathways, including proline biosynthesis, glutamine metabolism, uridine metabolism, histidine metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six abnormally expressed metabolic enzymes that are closely associated with the altered metabolic pathways were further discovered in esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5-carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of cancer metabolic reprogramming.

Integrated Genome Wide Association Study (GWAS) Identifies SNPs Associated with Outcome in Pediatric AML

Acute myeloid leukemia (AML) treatment response remains poorly understood. Although multiple studies have focused on understanding the transcriptomic and epigenetic landscape of AML, a genome-wide analysis of SNPs in pediatric AML has not yet been investigated in depth. Thus, we sought to identify genetic variants predictive of AML response, relapse, and survival in pediatric AML patients. For this study, we generated genome-wide SNP data patients (n=160) treated on the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) using Infinium Omni 2.5M Exome Beadchip. Standard GWAS QC procedure was followed in order to remove SNPs with call rate < 95%, monomorphic SNPs, SNPs with MAF<5% and samples with call rate<95%. Following QC, a risk-adjusted multi-outcome integrative GWAS was performed to identify SNPs associated with minimal residual disease (MRD) following induction I, relapse-free survival (RFS) and overall survival (OS). We performed a risk-adjusted analysis to identify 21 SNPs mapping to 14 genes at an endpoint-integrative p value <2x10-5. Table 1 provides list of genes with SNPs significantly associated with MRD, RFS, OS as well as in the integrated analysis at <2x10-5. Of interest multiple SNPs in DICER1, which is a key enzyme required for the biogenesis of microRNAs and small interfering RNAs were significantly associated with clinical outcome with promise integrated analysis at p = 0.000011, supported by associations with MRD, RFS and OS at p <0.002 (Figure 1A). DICER1 is over-expressed in AML with its expression under the influence of hematopoietic transcript factor, GATA1. RAI14, a retinoic acid induced 14 is a prognostic marker of poor response in solid tumors and has been associated with development of drug resistance. Multiple SNPs in RAI14 were significantly associated with clinical endpoints. Figure 1B shows RAI14 SNP rs336474 with C allele significantly associated with better RFS (p= 0.027) and OS (p=0.007), with an integrated p= 0.000004. SNP in upstream of RBFOX1, a RNA binding fox-1 homolog 1 and within intron of GRIN2A, glutamate ionotropic receptor NMDA type subunit 2A were significantly associated with MRD, RFS and OS (all p<0.005) and integrated p =0.00001 (Figure 1C). SNPs within genes involved in pyrimidine metabolism such as UPP2, a uridine phosphorylase; tumor suppressor genes such as JPH3, which codes for junctophilin; LILRB4 which encodes for a Leukocyte Immunoglobulin Like Receptor B4, that regulates inflammatory responses and cytotoxicity; HACE1 a potential tumor suppressor involved in the solid tumors pathophysiology; ANK2, an ankyrin family protein with role in cell proliferation and motility; BIRC8, which is implicated in CML disease progression etc. In conclusion, our results demonstrate significance of genome-wide investigation of SNPs to identify novel and clinically relevant SNPs of prognostic significance in childhood AML. We will present the in depth results of our integrated GWAS analysis as well as validation in independent patient cohorts. In summary, our results constitute one of the first integrated GWAS analyses to identify SNPs of prognostic significance in pediatric AML.Acknowledgments: We are thankful for funding from NIH R01-CA139246 and ALSAC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Vitamin D Regulation of the Uridine Phosphorylase 1 Gene and Uridine-Induced DNA Damage in Colon in African Americans and European Americans

African Americans have the greatest colorectal cancer (CRC) burden in the United States; interethnic differences in protective effects of vitamin D might contribute to disparities. 1α,25(OH)2D3 vitamin D (the active form of vitamin D) induces transcription of the uridine phosphorylase gene (UPP1) in colon tissues of European Americans but to a lesser extent in colon tissues of African Americans. UPP1-knockout mice have increased intestinal concentrations of uridine and Deoxyuridine triphosphate (dUTP), have increased uridine-induced DNA damage, and develop colon tumors. We studied 1α,25(OH)2D3 regulation of UPP1 and uridine-induced DNA damage in the colon and differences in these processes between African and European Americans. We quantified expression and activity of UPP1 in response to 1α,25(OH)2D3 in young adult mouse colonic cells, human CRC cells (LS174T), and organoids (derived from rectosigmoid biopsy samples of healthy individuals undergoing colonoscopies) using quantitative polymerase chain reaction, immunoblot, and immunocytochemistry assays. Binding of the vitamin D receptor to UPP1 was tested by chromatin immunoprecipitation. Uridine-induced DNA damage was measured by fragment-length analysis in repair enzyme assays. Allele-specific 1α,25(OH)2D3 responses were tested using luciferase assays. Vitamin D increased levels of UPP1 mRNA, protein, and enzymatic activity and increased vitamin D receptor binding to the UPP1 promoter in young adult mouse colonic cells, LS174T cells, and organoids. 1α,25(OH)2D3 significantly reduced levels of uridine and uridine-induced DNA damage in these cells, which required UPP1 expression. Organoids derived from colon tissues of African Americans expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 and had increased uridine-induced DNA damage compared with organoids derived from tissues of European Americans. Luciferase assays with the T allele of single nucleotide polymorphism rs28605337 near UPP1, which is found more frequently in African Americans than European Americans, expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 than assays without this variant. We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH)2D3. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

Computational quantum chemistry, molecular docking, and ADMET predictions of imidazole alkaloids of Pilocarpus microphyllus with schistosomicidal properties.

Schistosomiasis affects million people and its control is widely dependent on a single drug, praziquantel. Computational chemistry has led to the development of new tools that predict molecular properties related to pharmacological potential. We conducted a theoretical study of the imizadole alkaloids of Pilocarpus microphyllus (Rutaceae) with schistosomicidal properties. The molecules of epiisopiloturine, epiisopilosine, isopilosine, pilosine, and macaubine were evaluated using theory models (B3lyp/SDD, B3lyp/6-31+G(d,p), B3lyp/6-311++G(d,p)). Absorption, distribution, metabolization, excretion, and toxicity (ADMET) predictions were used to determine the pharmacokinetic and pharmacodynamic properties of the alkaloids. After optimization, the molecules were submitted to molecular docking calculations with the purine nucleoside phosphorylase, thioredoxin glutathione reductase, methylthioadenosine phosphorylase, arginase, uridine phosphorylase, Cathepsin B1 and histone deacetylase 8 enzymes, which are possible targets of Schistosoma mansoni. The results showed that B3lyp/6-311++G(d,p) was the optimal model to describe the properties studied. Thermodynamic analysis showed that epiisopiloturine and epiisopilosine were the most stable isomers; however, the epiisopilosine ligand achieved a superior interaction with the enzymes studied in the molecular docking experiments, which corroborated the results of previous experimental studies on schistosomiasis.

Structural and Functional Analysis of Pyrimidine Nucleoside Phosphorylases of the NP-I and NP-II Families in Complexes with 6-Methyluracil

The structure of bacterial uridine phosphorylase (UPh) belonging to the NP-I family in complex with 6-methyluracil was determined for the first time at 1.17 A resolution. The structural features of bacterial UPh from the bacterium Vibrio cholerae (VchUPh) responsible for selectivity toward 6-methyluracil acting as a pseudosubstrate were revealed. The repulsion between the hydrophilic hydroxyl group of the active-site residue Thr93 of VchUPh and the hydrophobic methyl group of 6-methyluracil prevents the oxygen atom O4' of the ribose moiety and the phosphate oxygen atom O3P of ribose 1-phosphate from forming hydrogen bonds with OG1_Thr93, which are essential for the enzymatic reaction. This, apparently, makes VchUPh inactive in the enzymatic synthesis of 6-methyluridine from 6-methyluracil. Hence, Thr93 is the residue, the modification of which will allow VchUPh to catalyze the biotechnologically important synthesis of 6-methyluridine from 6-methyluracil. Taking into account high structural homology of the functionally significant regions of bacterial UPhs, this conclusion is also true for other bacterial UPhs. It was demonstrated that bacterial thymidine phosphorylases of the NP-II family cannot bind 6-methyluracil in a proper conformation required for the catalysis because of a close contact between the 6-methyl group and Phe210.

Study of Structural-Functional Organization of Nucleoside Phosphorylases of Gammaproteobacteria. Special Aspects of Functioning of Uridine Phosphorylase Phosphate-Binding Site

Series of mutant genes of prokaryotic uridine phosphorylases (Shewanella oneidensis MR-1, Escherichia coli) were constructed, and the resulting strains-producers of the corresponding proteins were obtained. Proteins were purified, and their physicochemical and fermentative properties were studied. On the basis of the obtained data, the role of individual amino acid residues of the polypeptide chain of uridine phosphorylases in the formation and functioning of the phosphate-binding site in these proteins was shown. The assumption of independent binding of two substrates, ion of inorganic phosphate (Pi) and uridine (Urd), by nucleoside phosphorylases, was made.

Synthesis of 99mTc-Radiolabeled Uridine as a Potential Tumor Imaging Agent

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and biomembranes, was radiolabeled with 99mTc to obtain a potential tumor imaging agent. The maximal radiochemical yield of about 96.5%, as determined by paper chromatography and instant thin-layer chromatography, was reached under the following optimum conditions: 1 mg of uridine, 20 μg of SnCl2·2H2O as reducing agent, 20 mg of mannitol as a stabilizer, and pH 8. 99mTc-uridine is stable in vitro at room temperature for up to 6 h post labeling. The biodistrbution study in tumor-bearing mice shows high target-to-nontarget ratio. These results match with the high docking score of the complex on uridine phosphorylase enzyme. 99mTc-uridine shows promise as a tumor imaging agent.

Schistosoma mansoni purine and pyrimidine biosynthesis: structures and kinetic experiments in the search for the best therapeutic target.

Schistosoma mansoni is the etiological agent of schistosomiasis, a debilitating treatment neglected tropical disease that affects approximately 218 million people worldwide. Despite its importance, the treatment of schistosomiasis relies on a single drug, praziquantel. Some reports on the resistance of S. mansoni to this drug have stimulated efforts to develop new drugs to treat this disease. S. mansoni possesses all the same pyrimidine pathways (de novo, salvage and thymidylate cycles) as those of its host. The opposite scenario is true for purine metabolism, in which only the salvage pathway is present. These pathways have previously been proposed as potential drug targets. Using modern molecular biology techniques, large-scale study of these pathways has become possible; 24 genes have been studied, and several protein structures and kinetic parameters have been determined. Unique characteristics of schistosomal enzymes have been obtained, which show that this organism possesses two isoforms of uridine phosphorylase (UP), which share 92% of identity. However, only one isoform has a canonical function, whereas the second isoform is expressed through all life stages and does not have a known function. In addition, the methylthioadenosine phosphorylase (MTAP) is one of the enzymes responsible for the previously described adenosine phosphorylase activity, thus representing one main difference between S. mansoni and its host. The study of adenine phosphoribosyltransferase has revealed possible differential expression of the APRT gene in females. This result is consistent with those obtained for the experimental treatment of schistosomiasis in monkeys with the adenosine analog tubercidin, which eliminates the disease mainly in females. These important conclusions may aid in the development of new alternative drugs to treat schistosomiasis.

ATP alters protein folding and function of Escherichia coli uridine phosphorylase

Uridine phosphorylase is one of the critical enzymes in the pyrimidine salvage pathway. Cells regenerate uridine for nucleotide metabolism by incorporating uracil with ribose-1-phosphate with this enzyme. Recent studies indicate that Escherichia coli uridine phosphorylase is destabilized in the presence of ATP. However, the mechanism underlying the destabilization process and its influence on uridine phosphorylase function remain to be established. Here, we comprehensively investigated the effects of ATP on protein folding and function of Escherichia coli uridine phosphorylase. Our results demonstrate that ATP apparently decreases the stability of uridine phosphorylase in a concentration-dependent manner. Additionally, simply increasing the level of ATP led to a reduction of enzymatic activity to complete inhibition. Further studies showed that uridine phosphorylase accumulates as a partially unfolded state in the presence of ATP. Moreover, ATP specifically accelerated the unfolding rate of uridine phosphorylase with no observable effects on the refolding process. Our preliminary findings suggest that ATP can alter the protein folding and function of enzymes via apparent destabilization. This mechanism may be significant for proteins functioning under conditions of high levels of ATP, such as cancer cell environments.

Enzymatic synthesis of ribo- and 2′-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling

Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2′-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2′-Deoxyribonucleosides were prepared from 2′-deoxyinosine using the same methodology with minor modifications.