Xanthine

The uric acid/xanthine H(+) symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

The synthesis of nucleobases in natural environments, especially in interstellar molecular clouds, is the focus of a long-standing debate regarding prebiotic chemical evolution. Here we report the simultaneous detection of all three pyrimidine (cytosine, uracil and thymine) and three purine nucleobases (adenine, xanthine and hypoxanthine) in interstellar ice analogues composed of simple molecules including H2O, CO, NH3 and CH3OH after exposure to ultraviolet photons followed by thermal processes, that is, in conditions that simulate the chemical processes accompanying star formation from molecular clouds. Photolysis of primitive gas molecules at 10 K might be one of the key steps in the production of nucleobases. The present results strongly suggest that the evolution from molecular clouds to stars and planets provides a suitable environment for nucleobase synthesis in space.

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson-Crick base pair to junction J2. Xanthine inserts between a G-U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.

Purine metabolism and oxidative stress are increasingly recognized as important contributors to ischemic brain injury following acute ischemic stroke (AIS). Although uric acid has been extensively investigated because of its dual antioxidant and pro-oxidant properties, comparatively limited data exist regarding the prognostic relevance of upstream purine metabolites such as xanthine and hypoxanthine. The present study aimed to evaluate the association between serum uric acid, xanthine, and hypoxanthine concentrations and neurological severity and functional outcome following AIS. We performed a prospective observational cohort study including 143 consecutive patients admitted with acute ischemic stroke in a tertiary neurological center. Neurological severity was assessed using the National Institutes of Health Stroke Scale (NIHSS), while functional outcome was evaluated using the modified Rankin Scale (mRS) at discharge and at 3-month follow-up. Plasma xanthine and hypoxanthine concentrations were quantified using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. Distribution normality was assessed using Shapiro-Wilk and Kolmogorov-Smirnov testing. Comparative analyses were performed using Student's t-test, the Mann-Whitney U test, and the Kruskal-Wallis test, according to the distribution and measurement scale of the variables. Correlations were assessed using Spearman's rank correlation coefficient and visualized using a heatmap. Receiver operating characteristic (ROC) curve analysis and multivariable logistic regression models were additionally performed in order to evaluate the predictive performance of purine metabolism biomarkers for unfavorable functional outcome. Numerically lower serum uric acid concentrations were observed in patients with poorer functional outcomes at discharge and at 3 months, although these differences were not statistically significant. Hypoxanthine concentrations varied significantly according to admission NIHSS severity categories (p = 0.022), with lower values observed among patients presenting severe neurological deficits. Plasma xanthine concentrations demonstrated significant association with unfavorable long-term functional outcome (p = 0.033) and exhibited the highest predictive performance in ROC analysis (AUC = 0.614). In the adjusted multivariable logistic regression model, admission NIHSS remained the main independent predictor of unfavorable 3-month functional outcome, whereas xanthine and the other purine biomarkers did not retain statistically significant independent associations. Purine metabolism biomarkers showed exploratory associations with neurological severity and functional outcome, but the underlying biological mechanisms were not directly assessed. Simultaneous assessment of uric acid together with xanthine and hypoxanthine may provide additional insight into stroke severity and neurological recovery, supporting the potential role of purine metabolism profiling as a complementary biomarker approach in acute ischemic stroke.