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Periodontitis is associated with abnormal purine metabolism, which is manifested by increased uric acid in host blood and increased expression of the purine-degrading enzyme, xanthine oxidoreductase (XOR), in periodontal tissues. Both XOR and uric acid are pro-oxidative and pro-inflammatory mediators under pathological conditions. Animal studies have found that injection of uric acid promotes the progression of periodontitis and that febuxostat (an XOR inhibitor) improves tissue destruction in periodontitis. Therefore, blocking the source of uric acid may be a therapeutic strategy to control the progression of periodontitis. In this article, the rationality of XOR inhibitors as potential therapeutic drugs for periodontitis is reviewed. The literature review results suggest that XOR inhibitors show antioxidative, anti-inflammatory, and anti-osteoclastic effects, and XOR inhibitors show clinical efficacy in the treatment of infectious, inflammatory and osteolytic diseases. Although there is no direct evidence to support the finding that XOR inhibitors can ameliorate periodontal microecological dysbiosis, these drugs can modulate intestinal microflora dysbiosis, and there is indirect evidence to support a beneficial effect of XOR inhibitors on periodontal microecological dysbiosis. In conclusion, XOR inhibitors may be used as immunomodulators for the adjuvant treatment of periodontitis by inhibiting inflammation, oxidative stress and anti-osteoclast effects.

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Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all. [ABSTRACT FROM AUTHOR]

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes. [ABSTRACT FROM AUTHOR]

Caste-based reproductive division of labour in social insects is built on asymmetries in resource allocation within colonies. Kings and queens dominantly consume limited resources for reproduction, while non-reproductive castes such as workers and soldiers help reproductive castes. Studying the regulation of such asymmetries in resource allocation is crucial for understanding the maintenance of sociality in insects, although the molecular background is poorly understood. We focused on uric acid, which is reserved and used as a valuable nitrogen source in wood-eating termites. We found that king- and queen-specific degradation of uric acid contributes to reproduction in the subterranean termite Reticulitermes speratus. The urate oxidase gene (RsUAOX), which catalyses the first step of nitrogen recycling from stored uric acid, was highly expressed in mature kings and queens, and upregulated with differentiation into neotenic kings/queens. Suppression of uric acid degradation decreased the number of eggs laid per queen. Uric acid was shown to be provided by workers to reproductive castes. Our results suggest that the capacity to use nitrogen, which is essential for the protein synthesis required for reproduction, maintains colony cohesion expressed as the reproductive monopoly held by kings and queens. [ABSTRACT FROM AUTHOR]

Background: The department of biochemistry at a tertiary care teaching hospital had the first offline early clinical exposure (ECE) on gout in October 2021 in the aftermath of COVID 19. This paper is based on our experiences of conducting ECE according to Competency Based Medical Education in the classroom. Aim: To share our experiences of conducting ECE in a faculty constrained setting. Methods: This was a mixed methods study. A total of 187 students out of 200 were present on the day of ECE. A pre- and post-test of ten questions on nucleotide metabolism was administered to the students. The subject for the module was a 48-year-old farmer suffering from gout who kindly consented to be a part of the program which was coordinated with the orthopedics department. After the module, the students wrote their reflections in the logbook and also gave their written feedback. Results: The reflections revealed that they felt motivated to study the biochemical aspects of the disease as this helped them understand the clinical picture. The feedback revealed that they preferred having more such ECE sessions. The mean post-test and pre-test scores were 7.7 and 5.6, respectively. Conclusion: ECE improved the students' learning and motivated them to study the subject. The reflection and feedback by students demonstrate that the students were enthusiastic and motivated to participate in the ECE. The objective in disseminating our findings is to share the ECE experiences so that we can standardize the competencies and objectives of common clinical conditions in biochemistry. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an opportunistic protozoan parasite known to affect the human brain. The infection has been associated with an increased incidence of schizophrenia; however, the link between the two conditions remains unclear. This study aimed to compare the plasma metabolome of schizophrenia and non-schizophrenia subjects with or without Toxoplasma infection. Untargeted metabolomic profiling was carried out by liquid chromatography-mass spectrometry. Elevation of the α-hydroxyglutaric acid level and reduced adenosine monophosphate, inosine, hypoxanthine and xanthine were found in the subjects with either toxoplasmosis or schizophrenia alone. These results suggest that purine catabolism is a common metabolic alteration in Toxoplasma infection and schizophrenia. The roles of these metabolites on the pathogenesis of schizophrenia in relation to Toxoplasma infection warrant further studies. [ABSTRACT FROM AUTHOR]

Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all.

Ureides, the degraded products of purine catabolism in Arabidopsis , have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1 , Ataln , and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1 , Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth. • Ureides support Arabidopsis growth during carbon starvation. • Purine degradation mutants reduce biomass under carbon starvation. • Root-centric purine degradation in carbon stressed WT. • Ureides enhances biomass in WT and Atxdh1 mutant. [ABSTRACT FROM AUTHOR]

Purine degradation products have been shown to play roles in plant response to stresses such as drought, salinity, extended dark, nitrogen deficiency, and pathogen infection. In this study, we used Arabidopsis wild-type (WT) and an Atxdh1 -knockout mutant defective in xanthine dehydrogenase1 (XDH1) to examine the role of degraded purine metabolites in the responses to wounding or UV-C stress applied to the middle leaves of the plant. Wounding or UV-C stress in the mutant resulted in lower fresh-weight, increased senescence symptoms, and increased cell death compared to WT plants. In addition, WT plants exhibited lower levels of oxidative stress indicators, reactive oxygen species, and malondialdehyde in their leaves than the mutant. Notably, transcripts and proteins functioning in the purine degradation pathway were regulated in such a way that it led to enhanced ureide levels in WT leaves 24h after applying the UV-C or wound stress. However, different remobilization of the accumulated ureides was observed after 72h of stress. In plants treated with UV-C, the concentration of allantoin was highest in young leaves, whereas in wounded plants it was lowest in these leaves and instead accumulated mainly in the middle leaves that had been wounded. These results indicated that in WT plants treated with UV-C, ureides were remobilized from the lower older and damaged leaves to support young leaf growth during the recovery period from stress. After wounding, however, whilst some ureides were remobilized to the young leaves, more remained in the wounded middle leaves to function as antioxidants and/or healing agents. [ABSTRACT FROM AUTHOR]

Key message: Degradation of nitrogen-rich purines is tightly and oppositely regulated under drought and low nitrogen supply in bread wheat. Allantoin is a key target metabolite for improving nitrogen homeostasis under stress.Abstract: The metabolite allantoin is an intermediate of the catabolism of purines (components of nucleotides) and is known for its housekeeping role in nitrogen (N) recycling and also for its function in N transport and storage in nodulated legumes. Allantoin was also shown to differentially accumulate upon abiotic stress in a range of plant species but little is known about its role in cereals. To address this, purine catabolic pathway genes were identified in hexaploid bread wheat and their chromosomal location was experimentally validated. A comparative study of two Australian bread wheat genotypes revealed a highly significant increase of allantoin (up to 29-fold) under drought. In contrast, allantoin significantly decreased (up to 22-fold) in response to N deficiency. The observed changes were accompanied by transcriptional adjustment of key purine catabolic genes, suggesting that the recycling of purine-derived N is tightly regulated under stress. We propose opposite fates of allantoin in plants under stress: the accumulation of allantoin under drought circumvents its degradation to ammonium (NH4+) thereby preventing N losses. On the other hand, under N deficiency, increasing the NH4+ liberated via allantoin catabolism contributes towards the maintenance of N homeostasis. [ABSTRACT FROM AUTHOR]

Estrogen substances in the environment are increasing dramatically, which interfere with the normal hormone level of human body, lead to the disorder of endocrine system and even cancer. It is difficult to screen a large number of environmental estrogen substances by existing estrogen effect detection methods, and the results are often affected by many factors, thus the development of new method has become an urgent task. Electrochemical method is promising to reflect cell proliferation by tracking intracellular purine bases directly. In this study, the estrogen level in MCF-7 cells on multiwall carbon nanotubes modified glassy carbon electrode (MWCNTs/GCE) could be tracked simply and conveniently, and the estrogen effect of estradiol could be reflected by electrochemistry in time and dose-dependent manners. Electrochemical method displayed the best tolerance to culture factors, such as different cell densities, serum types, culture medium types and serum estrogen-free methods, which responsed to estrogen effect higher than MTT (about 40%) and cell counting methods (about 50%). Further Western blotting analysis showed that the estrogen effect of estradiol promoted purine catabolism and up-regulated guanine deaminase (GDA) and adenine deaminase (ADA) expression, the key enzymes of purine catabolism pathway, in a dose-dependent manner. The up-regulation of GDA and ADA led to the increase of intracellular guanine and xanthine, which enhanced the electrochemical signal derived from guanine and xanthine. [Display omitted] • Electrochemistry could track estrogen level in MCF-7 cells simply and conveniently. • Electrochemistry displayed excellent environment tolerance compared to other methods. • E2 estrogen effect up-regulated GDA and ADA expression in purine catabolism pathway. [ABSTRACT FROM AUTHOR]

Purines are abundant among organic nitrogen sources and have high nitrogen content. Accordingly, microorganisms have evolved different pathways to catabolize purines and their metabolic products such as allantoin. Enterobacteria from the genera Escherichia, Klebsiella and Salmonella have three such pathways. First, the HPX pathway, found in the genus Klebsiella and very close relatives, catabolizes purines during aerobic growth, extracting all four nitrogen atoms in the process. This pathway includes several known or predicted enzymes not previously observed in other purine catabolic pathways. Second, the ALL pathway, found in strains from all three species, catabolizes allantoin during anaerobic growth in a branched pathway that also includes glyoxylate assimilation. This allantoin fermentation pathway originally was characterized in a gram-positive bacterium, and therefore is widespread. Third, the XDH pathway, found in strains from Escherichia and Klebsiella spp., at present is ill-defined but likely includes enzymes to catabolize purines during anaerobic growth. Critically, this pathway may include an enzyme system for anaerobic urate catabolism, a phenomenon not previously described. Documenting such a pathway would overturn the long-held assumption that urate catabolism requires oxygen. Overall, this broad capability for purine catabolism during either aerobic or anaerobic growth suggests that purines and their metabolites contribute to enterobacterial fitness in a variety of environments., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Schizophrenia (SZ) is a biochemically complex disorder characterized by widespread defects in multiple metabolic pathways whose dynamic interactions, until recently, have been difficult to examine. Rather, evidence for these alterations has been collected piecemeal, limiting the potential to inform our understanding of the interactions amongst relevant biochemical pathways. We herein review perturbations in purine and neurotransmitter metabolism observed in early SZ using a metabolomic approach. Purine catabolism is an underappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. We have observed a homeostatic imbalance of purine catabolism in first-episode neuroleptic-naive patients with SZ (FENNS). Precursor and product relationships within purine pathways are tightly correlated. Although some of these correlations persist across disease or medication status, others appear to be lost among FENNS suggesting that steady formation of the antioxidant uric acid via purine catabolism is altered early in the course of illness. As is the case for within-pathway correlations, there are also significant cross-pathway correlations between respective purine and tryptophan pathway metabolites. By contrast, purine metabolites show significant cross-pathway correlation only with tyrosine, and not with its metabolites. Furthermore, several purine metabolites (uric acid, guanosine, or xanthine) are each significantly correlated with 5-hydroxyindoleacetic acid (5-HIAA) in healthy controls, but not in FENNS at baseline or 4-week after antipsychotic treatment. Taken together, the above findings suggest that purine catabolism strongly associates with the tryptophan pathways leading to serotonin (5-HT) and kynurenine metabolites. The Lack of a significant correlation between purine metabolites and 5-HIAA, suggests alterations in key 5-HT pathways that may both be modified by and contribute to oxidative stress via purine catabolism in FENNS.

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes. [ABSTRACT FROM AUTHOR]

Purine catabolism is regarded as a housekeeping function that remobilizes nitrogen for plant growth and development. However, emerging evidence suggests that certain purine metabolites might contribute to stress protection of plants. Here, we show that in Arabidopsis, the intermediary metabolite allantoin plays a role in abiotic stress tolerance via activation of abscisic acid ( ABA) metabolism. The aln loss-of-function of ALN, encoding allantoinase, results in increased allantoin accumulation, genome-wide up-regulation of stress-related genes and enhanced tolerance to drought-shock and osmotic stress in aln mutant seedlings. This phenotype is not caused by a general response to purine catabolism inhibition, but rather results from a specific effect of allantoin. Allantoin activates ABA production both through increased transcription of NCED3, encoding a key enzyme in ABA biosynthesis, and through post-translational activation via high-molecular-weight complex formation of BG1, a β-glucosidase hydrolysing glucose-conjugated ABA. Exogenous application of allantoin to wild-type plants also activates the two ABA-producing pathways that lead to ABA accumulation and stress-responsive gene expression, but this effect is abrogated in ABA-deficient and BG1-knockout mutants. We propose that purine catabolism functions not only in nitrogen metabolism, but also in stress tolerance by influencing ABA production, which is mediated by the possible regulatory action of allantoin. [ABSTRACT FROM AUTHOR]

Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in purine bases. In Klebsiella pneumoniae, the genes responsible for the assimilation of purine ring nitrogen are distributed in three separated clusters. We characterized the gene cluster involved in the metabolism of allantoate (genes KPN_01761 to KPN_01771). The functional assignments of HpxK, as an allantoate amidohydrolase, and of HpxU, as a regulator involved in the control of allantoate metabolism, were assessed experimentally. Gene hpxU encodes a repressor of the RpiR family that mediates the regulation of this system by allantoate. In this study, the binding of HpxU to the hpxF promoter and to the hpxU-hpxW intergenic regio containing the divergent promoter for these genes was evidenced by electrophoretic mobility shift assays. Allantoate released the HpxU repressor from its target operators whereas other purine intermediate metabolites, such as allantoin and oxamate, failed to induce complex dissociation. Sequence alignment of the four HpxU identified operators identified TGAA-Ns-TTCA as the consensus motif recognized by the HpxU repressor. [ABSTRACT FROM AUTHOR]

Xanthine Oxidase (XO; EC 1.3.22) as well as Xanthine Dehydrogenase (XDH; EC 1.17.1.4) are two enzymes responsible for the last steps of purine metabolism, hydroxylation of a wide variety pyrimidine, pterin, and aldehyde. There are numerous publications evidencing not directly about regulating role of the hypoxanthine/xanthine existence and its catabolism in the row of the purine metabolic pathway. Activitie of XO were evaluated based on the method introduced by Litwack et all [1]. Protein quantity was calculated based on the Bradford methos. We have used Student t-test as well as ONE-WAY-ANOVA. We have chosen the best condition for evaluation of XO activity in the homogenate. The activity of XO in the rat brain in the presence of different substrates was the following in comparison with the control (1.2104±0.0000, p<0.05): for histidine 2.2190±0.4707, in the presence of allopurinol-1.6138±0.2690, for riboflavin 1.6138±0.1345 (with allopurinol - 1.4651±0.8773), for adenosine - 1.6138±0.2017 (with allopurinol - 1.2776±0.1345), for desoxyadenosine - 2.2997±0.5245, (with allopurinol - 1.2507±0.0672). We concluded, that regulation of Xanthineoxidoreductase by feedback mechanism might suppress the catabolism of purines and serves as a basis for the initiation of cells proliferative processes. [ABSTRACT FROM AUTHOR]

Schizophrenia (SZ) is a biochemically complex disorder characterized by widespread defects in multiple metabolic pathways whose dynamic interactions, until recently, have been difficult to examine. Rather, evidence for these alterations has been collected piecemeal, limiting the potential to inform our understanding of the interactions amongst relevant biochemical pathways. We herein review perturbations in purine and neurotransmitter metabolism observed in early SZ using a metabolomic approach. Purine catabolism is an underappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. We have observed a homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with SZ (FENNS). Precursor and product relationships within purine pathways are tightly correlated. Although some of these correlations persist across disease or medication status, others appear to be lost among FENNS suggesting that steady formation of the antioxidant uric acid (UA) via purine catabolism is altered early in the course of illness. As is the case for within-pathway correlations, there are also significant cross-pathway correlations between respective purine and tryptophan (TRP) pathway metabolites. By contrast, purine metabolites show significant cross-pathway correlation only with tyrosine, and not with its metabolites. Furthermore, several purine metabolites (UA, guanosine, or xanthine) are each significantly correlated with 5-hydroxyindoleacetic acid (5-HIAA) in healthy controls, but not in FENNS at baseline or 4-week after antipsychotic treatment. Taken together, the above findings suggest that purine catabolism strongly associates with the TRP pathways leading to serotonin (5-hydroxytryptamine, 5-HT) and kynurenine metabolites. The lack of a significant correlation between purine metabolites and 5-HIAA, suggests alterations in key 5-HT pathways that may both be modified by and contribute to oxidative stress via purine catabolism in FENNS. [ABSTRACT FROM AUTHOR]

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic-clonic seizures and reduced the time spent in the generalized tonic-clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na, K-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease. [ABSTRACT FROM AUTHOR]

Abstract: The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU), which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, are unstable in solution and decay nonstereospecifically to allantoin. In addition, nonenzymatic racemization of allantoin has been shown to occur at physiological pH. Since the further breakdown of allantoin is catalyzed by allantoinase, an enzyme that is specific for (S)-allantoin, an allantoin racemase is necessary for complete and efficient catabolism of uric acid. In this work, we characterize the structure and activity of allantoin racemase from Klebsiella pneumoniae (KpHpxA). In addition to an unliganded structure solved using selenomethionyl single-wavelength anomalous dispersion, structures of C79S/C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are presented. These structures reveal several important features of the active site including an oxyanion hole and a polar binding pocket that interacts with the ureido tail of allantoin and serves to control the orientation of the hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-enantiomers of allantoin is demonstrated, and analysis of the steady-state kinetics of KpHpxA yielded a k cat/K m of 6.0×105 M−1 s−1. Mutation of either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this enzyme. The data presented provide new insights into the activity and substrate specificity of this enzyme and enable us to propose a mechanism for catalysis that is consistent with the two-base mechanism observed in other members of the aspartate/glutamate family. [Copyright &y& Elsevier]

Abstract: The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism. [Copyright &y& Elsevier]

Respiratory muscle weakness resulting from both diaphragmatic contractile dysfunction and atrophy has been hypothesized to contribute to the weaning difficulties associated with prolonged mechanical ventilation (MV). While it is clear that oxidative injury contributes to MV-induced diaphragmatic weakness, the source(s) of oxidants in the diaphragm during MV remain unknown. These experiments tested the hypothesis that xanthine oxidase (XO) contributes to MV-induced oxidant production in the rat diaphragm and that oxypurinol, a XO inhibitor, would attenuate MV-induced diaphragmatic oxidative stress, contractile dysfunction, and atrophy. Adult female Sprague-Dawley rats were randomly assigned to one of six experimental groups: 1) control, 2) control with oxypurinol, 3)12 h of MV, 4)12 h of MV with oxypurinol, 5) 18 h of MV, or 6)18 h of MV with oxypurinol. XO activity was significantly elevated in the diaphragm after MV, and oxypurinol administration inhibited this activity and provided protection against MV-induced oxidative stress and contractile dysfunction. Specifically, oxypurinol treatment partially attenuated both protein oxidation and lipid peroxidation in the diaphragm during MV. Further, XO inhibition retarded MV-induced diaphragmatic contractile dysfunction at stimulation frequencies >60 Hz. Collectively, these results suggest that oxidant production by XO contributes to MV-induced oxidative injury and contractile dysfunction in the diaphragm. Nonetheless, the failure of XO inhibition to completely prevent MV-induced diaphragmatic oxidative damage suggests that other sources of oxidant production are active in the diaphragm during prolonged MV. [ABSTRACT FROM AUTHOR]

Abstract: The adenine deaminase of A. nidulans, encoded by nadA, can be considered both as a catabolic and a purine salvage enzyme. We show that its transcriptional regulation reflects this double metabolic role. As all other genes involved in purine utilisation it is induced by uric acid, and this induction is mediated by the UaY transcription factor. However, it is also independently and synergistically induced by adenosine by a UaY-independent mechanism. At variance with all other enzymes of purine catabolism it is not repressed but induced by ammonium. This is at least partly due to the ammonium responsive GATA factor, AreA, acting in the nadA promoter as a competitor rather than in synergy with UaY. The adB gene, encoding adenylo-succinate synthetase, which can be considered both a biosynthetic and a salvage pathway enzyme, shares with nadA both ammonium and adenosine induction. [Copyright &y& Elsevier]

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato ( Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various 14C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [14C]formate, [2-14C]glycine and [2-14C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis. [ABSTRACT FROM AUTHOR]

The ureide pathway, which produces ureides from uric acid, is an essential purine catabolic process for storing and transporting the nitrogen fixed in leguminous plants and some bacteria. PucM from Bacillus subtilis was recently characterized and found to catalyze the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step. PucM has 121 amino acid residues and shows high sequence similarity to the functionally unrelated protein transthyretin (TrR), a thyroid hormone-binding protein. Therefore, PucM belongs to the TTR-related proteins (TRP) family. The crystal structures of PucM at 2.0 A and its complexes with the substrate analogs 8-azaxanthine and 5,6-diaminouracil reveal that even with their overall structure similarity, homotetrameric PucM and TFR are completely different, both in their electrostatic potential and in the size of the active sites located at the dimeric interface. Nevertheless, the absolutely conserved residues across the TRP family, including His-14, Arg-49, His-105, and the C-terminal Tyr-118-Arg-119-Gly-120-Ser-121, indeed form the active site of PucM. Based on the results of site-directed mutagenesis of these residues, we propose a possible mechanism for HIU hydrolysis. The PucM structure determined for the TRP family leads to the conclusion that diverse members of the TRP family would function similarly to PucM as HIU hydrolase. [ABSTRACT FROM AUTHOR]

In order to detect predicted changes in purine catabolism during the annual reproductive cycle of brown trout, we measured the activity of xanthine dehydrogenase, urate oxidase, and allantoinase. In kidney, only xanthine dehydrogenase was detected. In female liver and kidney, the activity of these enzymes was higher in May and decreased during vitellogenesis, with urate oxidase being undetectable in this period. In male liver, a similar variation pattern was found; however, in kidney, high activities were found in both May and December. These results suggest an influence of sex hormones in trout purine catabolism, especially in females. [ABSTRACT FROM AUTHOR]

A new gene ofNeurospora crassa, designatedpco-1, was characterized and shown to regulate the expression of several genes which encode enzymes required for the catabolism of purines. Unlike the wild type, apco-1mutant created by repeat-induced point mutation cannot utilize purines as a nitrogen source. The PCO1 protein contains a Zn(II)2Cys6 binuclear cluster motif near its N-terminus, followed by a putative coiled-coil motif. A chemical crosslinking experiment demonstrated that PCO1 forms homodimers. PCO1 binds to CGG-N6-CCG elements located in the upstream promoter region of four genes encoding purine catabolic enzymes. Northern blot analysis demonstrated that a functional PCO1 protein is required for induction ofxdh, which encodes xanthine dehydrogenase. Moreover, PCO1 was required for induction of three different purine catabolic enzymes. Two glutamine-rich domains occur in the C-terminal region of PCO1 and at least one of the glutamine-rich regions is required for PCO1 function, suggesting that they might play a role in transcriptional activation. The PCO1 protein does not interact with the global-acting NIT2 protein or the negative-acting NMR protein that functions in nitrogen catabolite repression. Induction of thexdhgene and synthesis of xanthine dehydrogenase is completely dependent upon PCO1, but does not require the global-acting NIT2 protein, suggesting that it is controlled by a novel regulatory mechanism. [ABSTRACT FROM AUTHOR]

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity. [ABSTRACT FROM AUTHOR]

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an Mr of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, 59Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Michaelian kinetics typical for a bisubstrate enzyme with apparent Km values for NAD+, hypoxanthine and xanthine of 35, 160 and 70 μM, respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-free media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine. [ABSTRACT FROM AUTHOR]

Intensified adenosine triphosphate (ATP) degradation following therapeutic hyperthermia is often observed in solid tumors. As a result, accumulation of purine catabolites can be expected together with formation of protons at several stages during degradation to the final product, uric acid. Proton formation in turn can contribute to the development of heat-induced acidosis. Furthermore, oxidation of hypoxanthine and xanthine may result in generation of reactive oxygen species, which may lead to DNA damage, lipid peroxidation and protein denaturation, thus also contributing to heat-induced cytotoxicity. In hyperthermia experiments a tumor-size-dependent, significant increase in the levels of the following catabolites has been demonstrated: Σ[IMP+GMP] (sum of guanosine and inosine monophosphate levels), inosine, hypoxanthine, xanthine and uric acid, along with a drop in ATP and guanosine triphosphate (GTP) levels. These data suggest that formation of reactive oxygen species and protons during purine degradation may indeed play a significant role in the antitumor effect of hyperthermia. [ABSTRACT FROM AUTHOR]

The purine catabolic enzymes uricase, allantoinase, allantoicase and ureidoglycollase are highly induced by purines in wild-type Schizosaccharomyces pombe cells. In contrast, urease activity is constitutive. Attempts have been made to identify the inducer or inducers involved. There appears to be a sequential form of induction, uricase being induced by uric acid, allantoinase by allantoic acid (and possibly allantoin), allantoicase by allantoic acid, ureidoglycollase by ureidoglycollic acid. This sequential control of purine breakdown is compared with that found in other fungi. [ABSTRACT FROM AUTHOR]

Purines such as hypoxanthine, xanthine, uric acid, allantoin and allantoic acid serve as sole nitrogen sources for the yeast Schizosaccharomyces pombe. A number of classes of mutants unable to use purines have been isolated and genetically analysed. Mutants in the urol gene lack uricase, all1 lack allantoinase, ala1 lack allantoicase whilst in ure1, ure2, ure3 and ure4 genes lack urease activity. Mutants in four hyp genes are unable to convert hypoxanthine to uric acid whilst mutation in xan1 results in impaired growth with xanthine. hyp5 strains are unable to convert both hypoxanthine and xanthine to uric acid. The mutations are recessive and none of the loci are linked to each other. The possible catalytic steps involved are discussed. [ABSTRACT FROM AUTHOR]

High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. Here, we show that high sugar induces dehydration in adult Drosophila , and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. Our model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target. • Sugar-induced lifespan shortening can be uncoupled from obesity and insulin resistance • A high-sugar diet induces water imbalance in adult Drosophila • High-sugar feeding shortens fly lifespan through dysregulation of purine catabolism • Dietary sugar intake in humans is associated with circulating purine levels van Dam et al. disentangle the physiological effects of high-sugar feeding on survival using adult Drosophila. They uncouple diet-induced obesity and insulin resistance from shortened lifespan by rescuing sugar-induced water imbalance. Dietary sugar is linked to kidney dysfunction and dysregulation of purine catabolism in both flies and humans, emphasizing this pathway as a promising target for potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Nitrogen (N) deficiency is a main environmental factor that induces early senescence. Cotyledons provide an important N source during germination and early seedling development. In this study, we observed that N deficient condition enhanced gene expression involved in purine catabolism in cotyledons of Chinese cabbage ( Brassica rapa ssp. Pekinensis ). Seedlings grown with added allopurinol, an inhibitor of xanthine dehydrogenase, in the growth medium showed reduced chlorophyll degradation in cotyledons and lower fresh weight, compared with seedlings grown on normal medium. On the basis of these results, we speculated that xanthine-derived metabolites might affect both seedling growth and early senescence in cotyledons. To confirm this, seedlings were grown with exogenous xanthine to analyze the role of xanthine-derived metabolites under N deficient condition. Seedlings with xanthine as the sole N-source grew faster, and more cotyledon chlorophyll was broken down, compared with seedlings grown without xanthine. The expression levels of senescence- and purine metabolism-related genes in cotyledons were higher than those in seedlings grown without xanthine. These results indicate the possibility that xanthine plays a role as an activator in both purine catabolism and chlorophyll degradation in cotyledons under N deficient condition.

High-calorie diet, circadian rhythms and metabolic features are intimately linked. However, the mediator(s) between nutritional status, circadian rhythms and metabolism remain largely unknown. This article aims to clarify the key metabolic pathways bridging nutritional status and circadian rhythms based on a combination of metabolomics and molecular biological techniques. A mouse model of high-fat diet-induced obesity was established and serum samples were collected in obese and normal mice at different zeitgeber times. Gas chromatography/mass spectrometry, multivariate/univariate data analyses and metabolic pathway analysis were used to reveal changes in metabolism. Metabolites involved in the metabolism of purines, carbohydrates, fatty acids and amino acids were markedly perturbed in accordance with circadian related variations, among which purine catabolism showed a typical oscillation. What's more, the rhythmicity of purine catabolism dampened in the high-fat diet group. The expressions of clock genes and metabolic enzymes in the liver were measured. The mRNA expression of Xanthine oxidase (Xor) was highly correlated with the rhythmicity of Clock, Rev-erbα and Bmal1, as well as the metabolites involved in purine catabolism. These data showed that a high-fat diet altered the circadian rhythm of metabolic pathways, especially purine catabolism. It had an obvious circadian oscillation and a high-fat diet dampened its circadian rhythmicity. It was suggested that circadian rhythmicity of purine catabolism is related to circadian oscillations of expression of Xor, Uox and corresponding clock genes. [ABSTRACT FROM AUTHOR]

Metabolism of purine bases remains poorly understood in the pathogenic bacterium Mycobacterium tuberculosis and closely related, nonpathogenic Mycobacterium smegmatis (Msm). To gain insight into the purine metabolism in mycobacteria, we tested uptake of purine bases with a ΔpurF Msm mutant with an inactive purine de novo biosynthesis pathway and confirmed that hypoxanthine and guanine, but not xanthine, can serve as nucleotide precursors for recycling in the salvage pathway. Further, we focused on purine catabolism in wild-type (wt) Msm. We found that only xanthine and guanine could serve as a sole nitrogen source for wt Msm. These data confirm that Msm catabolism of purines is directed mainly via oxidative guanine to xanthine interconversion and not through metabolic conversion of hypoxanthine to xanthine. Our data represent the first experimental evidence confirming the use of 8-oxo-purines as a nitrogen source by Msm., (Copyright © 2019 Elsevier Ltd. All rights reserved.)