Uridine phosphorylase

Weaver, K., Chen, D., Walton, L., Elwell, L., and Ray, R, Uridine phosphorylase purified from total crude extracts of E. coli using Q Sepharose and radial-flow chromatography, Biopharmacology, 37, 25, 1990. 

Like many reported A -transglycosylations, this reaction uses uncharacterized nucleoside phosphorylases from whole cells held at 50-60 °C, a temperature well above the range for viability of the parent microorganism. Remarkable temperature stability has been reported for three well-known NPs of E. coli purine nucleoside phosphorylase (PNP), uridine phosphorylase (URDP) and thymidine phosphorylase. ... 

Fig. lA. Anabolic and catabolic pathways of 5-FU. DPD dihydropyrimidine dehydrogenase, DP di-hydropyrimidinase, pUP beta-ureidopropionase, UP uridine phosphorylase, OPRT orotate phospho-ribosyl transferase, UK uridine kinase, TP thymidine phosphorylase, TK thymidine kinase, RNR ribonucleotide reductase. The three active metabolites (shown in rectangles) are FdUMP (5-fluoro-2 -deoxyuridine 5 -monophosphate) inhibiting TS (thymidylate synthase), and FUTP (5-fluorouridine 5 -triphosphate) and FdUTP (5-fluoro 2 -deoxyuridine 5 -triphosphate) interfering with RNA and DNA, respectively. 

Pyrimidine nucleosides and their analogs as uridine phosphorylase inhibitors and potential chemotherapeutic agents 91CLY171. 

A close look at this reaction reveals that in the opposite direction, the reaction is of the phosphorolysis type. For this reason, the enzymes catalyzing the reaction with ribose-l-phosphate are called phosphorylases, and they also participate in nucleic acid degradation pathways. Purine nucleoside phosphorylases thus convert hypoxanthine and guanine to either inosine and guanosine if ribose-l-phosphate is the substrate or to deoxyinosine and deoxyguanosine if deoxyribose-1-phosphate is the substrate. Uridine phosphorylase converts uracil to uridine in the presence of ribose-l-phosphate, and thymidine is formed from thymine and deoxyribose-l-phosphate through the action of thymidine phosphorylase. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Recombinant uridine phosphorylase from E. coli lysate [9]... 

The XLL cross-axis CPC, with a 250-mL capacity column, was used for the purification of recombinant enzymes such as purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UrdPase) from a crude Escherichia coli lysate. The polymer-phase system used in these separations was 16% (w/w) PEG 1000-12.5% (w/w) potassium phosphate at pH 6.8. The separation was performed at 750 rpm at a flow rate of 0.5 mL/min using the upper phase as a mobile phase. About 1.0 mL of crude lysate, containing PNP in 10 mL of the above solvent system, was loaded into the multilayer coil. Purified PNP was harvested in 45-mL fractions. The SDS-PAGE analysis clearly demonstrated that PNP was highly purified in a one-step elution with the XLL cross-axis CPC. 

Recombinant uridine phosphorylase [8] SDS-PAGE Enzymatic activity by Magni method... 

Enterobacteria aerogenes can be grown when dimethyl phosphate is the sole source of phosphorus, and under these conditions a phosphodiesterase can be isolated from the micro-organism which utilizes dialkyl esters of phosphoric acid as substrates. Phosphodiesterases which have been isolated hitherto utilize nucleoside 2, 3 - or 3, 5 -phosphates and 4-nitrophenyl esters as substrates. A studyhas confirmed the hypothesis that phosphate binds to uridine phosphorylase before uridine, and that uracil leaves the enzyme before ribose-1 -phosphate. 

Ftorafur) fluorouracil by thymidine/uridine phosphorylase in the liver Uracil Substrate for dihydropyridimidine dehydrogenase (DPD) inhibits degradation of fluorouracil rash, and neurotoxicity (dizziness, confusion, ataxia) ... 

For example, S. enterica subsp enterica and S. bongori contain a conserved chromosomal cluster encoding an RNm transporter PnuC and a paralogue of uridine phosphorylase (Udp), which may be considered a candidate for the role of bacterial RNMPSE. 

Consistent with the metabolic data, there is no dihydrofolate reductase/thymidylate synthase activity (75). Thymidine is salvaged by a phosphotransferase. Uracil PRTase, uridine phosphorylase, cytidine deaminase and uridine and cytidine phosphotransferases were found. The major pyrimidine salvaged is uracil via its PRTase. The lack of incorporation of salvaged uracil into DNA and the lack of thymidylate synthase indicates that this parasite cannot synthesize TMP from UMP. It is dependent on salvage for its thymidine requirements. This parasite possesses a hydroxyurea-resistant ribonucleotide reductase and can synthesize deoxycytidine nucleotides from cytidine nucleotides. 

The salvage activities of T. foetus and T. vaginalis also differ (22,77). Unlike T. foetus, the level of uracil PRTase activity is very low. Uracil is converted into uridine by a uridine phosphorylase uridine is then phosphorylated by a uridine phosphotransferase to UMP (Fig. 6.15). Cytidine and thymidine also are converted into their nucleotide monophosphates by phosphotransferase activities. There is no detectable pyrimidine nucleoside kinase activity and the only significant interconversion among salvaged pyrimidines is catalyzed by cytidine deaminase to form uridine. 

Pyrimidine phosphorylase can use all of the pyrimidines but has a preference for uracil and is sometimes called uridine phosphorylase. The phosphorylase uses cytosine fairly well but has a very, very low affinity for thymine therefore, a ribonucle-oside containing thymine is almost never made in vivo. A second phosphorylase, thymine phosphorylase, has a much higher affinity for thymine and adds a deoxyri-bose residue (see Fig. 41.17). 

The aminodeoxy analogs were found to be very potent inhibitors of uridine phosphorylase isolated from sarcoma, and they exhibited no apparent cytotoxicity against sarcoma 180 host cells. Furthermore, they have shown excellent water solubility, which is a factor critical for the formulation that often limits the usefulness of a particular compound as a chemotherapeutic agent (85JMC971). 

Bock, K. Pedersen, C. Reaction of sugar esters with hydrogen fluoride. X. Derivatives of D-glucofuranose and D-mannofuranose. Acta Chem. Scand. 1972, 26, 2360-2366. Ajmera, S. Bapat, A.R. Stephanian, E. Danenherg, P.V. Synthesis and interaction with uridine phosphorylase of 5 -deoxy-4, 5-difluorouridine, a new prodrug of 5-fluorouracyl. J. Med. Chem. 1988, 31, 1094-1098. 

HSV-thymidine kinase [721], uridine phosphorylase [669], and xanthine oxidase [668, 722]... 

Although cross-axis CPCs yield less efficient separations than the type-J multilayer CPC, they provide more stable retention of the stationary phase and are therefore useful for large-scale preparative separations with polar solvent systems. These are especially useful for the purification of proteins with aqueous-aqueous polymer phase systems composed of PEG and potassium phosphate. The crossaxis CPC has been used for the purification of various enzymes including choline esterase, ketosteroid isomerase, purine nucleoside phosphorylase, lactic acid dehydrogenase, uridine phosphorylase (see Proteins Cross-Axis Coil Planet Centrifuge Separation, p. 1935). 

---