Role of phosphate-coordinating arginine residues in the thermal stability of uridine phosphorylase from Shewanella oneidensis MR-1.

The role of phosphate-coordinating arginine residues in the thermal stability of uridine phosphorylase from Shewanella oneidensis MR-1 was investigated by mutation analysis. Uridine phosphorylase mutant genes were constructed by site-directed mutagenesis. The enzyme mutants were prepared and isolated, and their kinetic parameters were determined. It was shown that all these arginine residues play an important role both in the catalysis and thermal stability. The arginine residues 176 were demonstrated to form a kind of a phosphate pore in the hexameric structure of uridine phosphorylase, and they not only contribute to thermal stabilization of the enzyme but also have a regulatory function. The replacement of arginine 176 with an alanine residue resulted in a significant decrease in the kinetic stability of the enzyme but led to a twofold increase in its specific activity.

Metabolites modulate the functional state of human uridine phosphorylase I.

Metabolic pathways in cancer cells typically become reprogrammed to support unconstrained proliferation. These abnormal metabolic states are often accompanied by accumulation of high concentrations of ATP in the cytosol, a phenomenon known as the Warburg Effect. However, how high concentrations of ATP relate to the functional state of proteins is poorly understood. Here, we comprehensively studied the influence of ATP levels on the functional state of the human enzyme, uridine phosphorylase I (hUP1), which is responsible for activating the chemotherapeutic pro-drug, 5-fluorouracil. We found that elevated levels of ATP decrease the stability of hUP1, leading to the loss of its proper folding and function. We further showed that the concentration of hUP1 exerts a critical influence on this ATP-induced destabilizing effect. In addition, we found that ATP interacts with hUP1 through a partially unfolded state and accelerates the rate of hUP1 unfolding. Interestingly, some structurally similar metabolites showed similar destabilization effects on hUP1. Our findings suggest that metabolites can alter the folding and function of a human protein, hUP1, through protein destabilization. This phenomenon may be relevant in studying the functions of proteins that exist in the specific metabolic environment of a cancer cell.

Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici.

Uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role. Here, we biochemically and structurally characterized two evolutionarily divergent uridine phosphorylases, PcUP1 and PcUP2 from the oomycete pathogen Phytophthora capsici. Our analysis of other oomycete genomes revealed that both uridine phosphorylases are present in Phytophthora and Pythium genomes, but only UP2 is seen in Saprolegnia spp. which are basal members of the oomycetes. Moreover, uridine phosphorylases are not found in obligate oomycete pathogens such as Hyaloperonospora arabidopsidis and Albugo spp. PcUP1 and PcUP2 are upregulated 300 and 500 fold respectively, within 90 min after infection of pepper leaves. The crystal structures of PcUP1 in ligand-free and in complex with uracil/ribose-1-phosphate, 2'-deoxyuridine/phosphate and thymidine/phosphate were analyzed. Crystal structure of this uridine phosphorylase showed strict conservation of key residues in the binding pocket. Structure analysis of PcUP1 with bound ligands, and site-directed mutagenesis of key residues provide additional support for the "push-pull" model of catalysis. Our study highlights the importance of pyrimidine salvage during the earliest stages of infection.

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.

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis.

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2'-, 3'- and 5'-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2'- or 5'-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2'- and 5'-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3'-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2'-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.

Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Šresolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

Analysis of two Schistosoma mansoni uridine phosphorylases isoforms suggests the emergence of a protein with a non-canonical function.

Reports of Schistosoma mansoni strains resistant to praziquantel, the only therapeutic strategy available for the treatment of schistosomiasis, have motivated the scientific community towards the search for new possible therapies. Biochemical characterization of the parasite's metabolism is an essential component for the rational development of new therapeutic alternatives. One of the so far uncharacterized enzymes is uridine phosphorylase (UP) (EC 2.4.2.3), for which the parasite genome presents two isoforms (SmUPa and SmUPb) that share 92% sequence identity. In this paper, we present crystal structures for SmUPa and SmUPb in their free states as well as bound to different ligands. This we have complemented by enzyme kinetic characterization and phylogenetic analyses. Both enzymes present an overall fold and active site structure similar to other known UPs. The kinetic analyses showed conclusively that SmUPa is a regular uridine phosphorylase but by contrast SmUPb presented no detectable activity. This is particularly noteworthy given the high level of sequence identity between the two isoforms and is probably the result of the significant differences observed for SmUPb in the vicinity of the active site itself, suggesting that it is not a UP at all. On the other hand, it was not possible to identify an alternative function for SmUPb, although our phylogenetic analyses and expression data suggest that SmUPb is still functional and plays a role in parasite metabolism. The unusual UPb isoform may open up new opportunities for understanding unique features of S. mansoni metabolism.

High-syn conformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures of Shewanella oneidensis MR-1 uridine phosphorylase in the free form and in complex with uridine.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP from Shewanella oneidensis MR-1 (SoUP) was performed in Escherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-syn conformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

Crystallization and preliminary X-ray study of Vibrio cholerae uridine phosphorylase in complex with 6-methyluracil.

Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh-6-MU complex at atomic resolution are reported.