Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

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.     

A re-evaluation of the tissue distribution and physiology of xanthine oxidoreductase

Summary Xanthine oxidoreductase is an enzyme which has the unusual property that it can exist in a dehydrogenase form which uses NAD⁺ and an oxidase form which uses oxygen as electron acceptor. Both forms have a high affinity for hypoxanthine and xanthine as substrates. In addition, conversion of one form to the other may occur under different conditions. The exact function of the enzyme is still unknown but it seems to play a role in purine catabolism, detoxification of xenobiotics and antioxidant capacity by producing urate. The oxidase form produces reactive oxygen species and, therefore, the enzyme is thought to be involved in various pathological processes such as tissue injury due to ischaemia followed by reperfusion, but its role is still a matter of debate. The present review summarizes information that has become available about the enzyme. Interpretations of contradictory findings are presented in order to reduce confusion that still exists with respect to the role of this enzyme in physiology and pathology.     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity,oxidation–reduction potentials of its redox-active centers,and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD⁺ as the preferred electron acceptor. In the hypoxanthine:NAD⁺ assay, the specific activity of purified XDH was 26.7 U (mg protein)⁻¹. Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD⁺. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an α₄β₄ structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.     

The Involvement of Cyclic ADPR in Photoperiodic Flower Induction of Pharbitis nil

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from NAD⁺ by ADP-ribosyl cyclases described for several animal cells. Pharmacological studies suggest that cADPR is an endogenous modulator of Ca²⁺-induced Ca²⁺ release channels. There is also information about the sub-micromolar concentration of cADPR in plant cells. Whether cADPR can act as a Ca²⁺-mobilizing intracellular messenger in plant tissue is an unresolved question. Despite the obvious importance of monitoring cADPR cellular levels under various physiological conditions in plants, its measurement has been technically difficult and requires specialized reagents. In the present study a widely applicable sensitivity assay for cADPR is described. We show that Pharbitis nil tissue from cotyledons contains a certain cADPR level. To explain the possible roles of this second messenger in photoperiodic flower induction, some physiological experiments were also performed. The exogenous applications of cADPR to Pharbitis nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. Nevertheless 8-Br-cADPR inhibited flowering when these compounds were applied during a 16-h-long inductive night. The effect of ruthenium red, a calcium channel blocker and ryanodine, a calcium channel stimulator, on the photoperiodic induction of flowering was also studied. Ruthenium red, when applied before and during an inductive 16-h dark period, slightly inhibited flowering, whereas ryanodine, when applied before and during a 12-h long subinductive night, stimulated flower bud formation. We also confirmed evidence that Ca²⁺ ions are involved in the photoperiodic induction of flowering. Thus, the obtained results may suggest the involvement of cyclic ADPR-activated Ca²⁺ mobilization in the photoperiodic flower induction process in Pharbitis nil.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

Catabolism of adenine derivatives in leaves: study of the role of light on the in vivo activity of xanthine dehydrogenase

The in vivo activity of xanthine dehydrogenase (E.C. 1.2.1.37) was followed in leaf discs excised from illuminated or darkened plants. In cotyledons of Pharbitis nil, 24 hours of darkness enhanced the in vivo activity of xanthine dehydrogenase which increased between 2 to 5-fold depending on the concentration of hypoxanthine of the solution where cotyledon discs were incubated. The same effect occurred in leaves of several other species, in plants with both high and low ureide content. However, the effect of light was not observed in leaves of Zea mays, Pennisetum americanum and Atriplex spongiosa, whereas, it appeared very clearly in other C₄ plants such as Sorghum sudanense and Portulaca oleracea. This enzymic activity in chlorophyll-deficient tobacco leaves was the same both for illuminated and darkened plants. In addition, the in vivo activity of xanthine dehydrogenase in roots of Pharbitis nil was not dependent upon the light conditions applied to leaves. In cotyledons of Pharbitis nil, the level of the in vivo activity of xanthine dehydrogenase was influenced by the energy of light and the duration of illumination. The supply of carbohydrates to darkened cotyledons had the same effect as light on the in vivo activity of xanthine dehydrogenase. It is proposed that the effect of light on the in vivo activity of xanthine dehydrogenase in leaves is mainly due to the production of photosynthates which changes the osmotic state of leaf tissue and thus modifies the level of the in vivo activity of xanthine dehydrogenase.     

Thermostable Xanthine Oxidase Activity from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t_1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg²⁺, Ag⁺ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.     

Formation of xanthine and the use of purine metabolites as a nitrogen source in Arabidopsis plants

In our recent paper in The Plant Journal,¹ we described the remobilization of purine metabolites during natural and dark induced senescence in wild type and Atxdh1 mutant lines impaired in xanthine dehydrogenase (XDH), a pivotal enzyme in the purine catabolism pathway. In the light of these observations and additional evidence shown here, we discuss the probable pathways leading to xanthine synthesis in Arabidopsis plants during senescence and the role that purine metabolites play as an ongoing source of nitrogen in plant growth.Key words: hypoxanthine, purine catabolism, senescence, xanthine, xanthine dehydrogenaseIn mammalian purine catabolism, hypoxanthine is oxidized to xanthine by xanthine oxidase.² In planta, xanthine can be synthesized in the purine degradation pathway, via three alternative precursors, guanine, xanthosine or hypoxanthine³ (Fig. 1A). Thus, the exact pathway leading to xanthine may depend on the species examined, the particular plant organ, developmental stage or specific environmental stimuli. For example, guanine and guanosine were shown to be the main precursor of ureides and CO₂ in cacao leaves⁴ while in tea leaves, elevated amounts of labeled xanthosine were recovered as ureides.^5,6 However, when hypoxanthine was used as a substrate for inosine monophosphate (IMP) formation in tobacco protoplasts⁷ more than 90% of labeled hypoxanthine was recovered as salvage products, nucleotides and RNA and only less then 10% was found as ureides in cacao leaves.⁴ Furthermore, when [8-¹⁴C]-hypoxanthine is supplied to soybean embryo axes or Jerusalem artichoke shoots it selectively labelled the guanine nucleotide pool.^3,8,9 These data do not support the possibility of hypoxanthine being a direct precursor for xanthine formation and illustrate the concept of species dependent differences in xanthine biosynthesis.¹⁰Open in a separate windowFigure 1Purine catabolism, xanthine and hypoxanthine accumulation and Arabidopsis plants growth. (A) Purine nucleotide catabolism in plants. Enzymes shown are: (1) AMP deaminase (EC 3.5.4.6), (2) IMP dehydrogenase (EC 1.1.1.205), (3) GMP synthase (EC 6.3.5.2), (4) 5''-nucleotidase, (5) Nucloeside phosphotransferase (EC 2.7.1.77), (6) Inosine-guanosine nucleosidase (EC 3.2.2.2), (7) Guanine deaminase (EC 3.5.4.15), (8) Xanthine dehydrogenase (EC 1.1.1.204), (9) Uricase EC 1.7.3.3, (10) Hydroxyisourate hydrolase (EC 3.5.2.17),1,18 (11) Allantoinase, allantoin amidohydrolase (EC 3.5.2.5), (12) Allantoicase, allantoate amidohydrolase (EC 3.5.3.4), (13) Ureidoglycolate lyase (EC 4.3.2.3), (14) Urease EC 3.5.1.5, (15) Allantoin deaminase (EC 3.5.3.9), (16) Ureidoglycine amidohydrolase (EC 3.5.3.-), (17) Ureidoglycolate hydrolase (EC 3.5.3.19). (B) Analysis of the purine metabolites, hypoxanthine and xanthine, in response to dark stress. Hypoxanthine and xanthine were determined by HPLC¹ in rosette leaves of wild-type (Col) and Ri14, XDH1 RNA interference plants after being kept in dark for 6 days and transferred to a 16-h light/8-h dark regime for recovery over an additional 3 days. Values are means ± SEM (n = 3). (C) Wild-type (Col) and XDH-compromised plants (KO, SALK_148364; Ri, XDH1 RNA interference) were germinated on ¼ MS medium and transplanted on the 5 day to a full MS medium (upper panel) or MS medium with 5.0 mM xanthine and urea as the sole nitrogen source. After transplanting the seedlings were left to grow for 14 days under a 16-h light/8-h dark regime (100 µmol m⁻² sec⁻¹) and then photographed. Leaf size was estimated using ImageJ software (http://rsb.info.nih.gov/ij/). Values are means ± SEM (n = 3).To study the possible role of hypoxanthine in xanthine formation in Arabidopsis we utilized XDH1 mutants. The mutants do not show any detectable XDH activity in-vitro when using hypoxanthine and/or xanthine as substrates.^1,11 Furthermore, no other enzyme is known to catalyze the conversion of hypoxanthine to xanthine, other than the molybdenum cofactor containing-XDH1. Yet, xanthine accumulation was readily detected in mutant leaves and was up to 100-fold higher than hypoxanthine either in normal growth conditions (Fig. 1B, time 0) or when exposed to dark induced senescence and to a light recovery period thereafter (Fig. 1B). These results indicate that most likely, hypoxanthine is not a major direct source for xanthine formation in Arabidopsis. The results imply that xanthosine or guanine are a source, although, one cannot exclude the possibility that hypoxanthine could be converted to xanthine in a pathway leading to inosine, IMP and then either via guanine or xanthosine, back to xanthine as illustrated in Figure 1A.In legumes inoculated with rhizobia, nitrogen is fixed initially as NH₃/NH₄⁺ that is subsequently incorporated through the purine pathway to form IMP, and finally ureides. The central role of purine catabolism in plant nitrogen metabolism was demonstrated mainly in legumes in which the purine nucleotides are degraded via uric acid and allantoin to urea and then to CO₂ and NH₃, which is then re-assimilated via the glutamine oxoglutarate aminotransferase (GOGAT) pathway (reviewed in ref. ³). What then is the role of purine catabolism pathways in non leguminous plants? Are the nitrogenous products of the degraded purines re-assimilated in non-legumes as in legume plants? We recently showed in Arabidopsis that a marked transition from assimilation, during the plants normal growth, to a state of rapid metabolite turnover occurs when plants were exposed to extended dark stress, senescence or even during normal diurnal cycles.¹⁰ This was depicted by the acceleration of purine catabolic recycling activities in which XDH1 plays a central role.¹ To test for a possible role of the accumulating purines as a source of nitrogen metabolites, we grew wild-type Arabidopsis plants and their XDH1 mutants under heterotrophic conditions. The agar plates contained either full MS nutrient solution with nitrate and ammonia or the purine metabolites, hypoxanthine (data not shown), xanthine or urea (Fig. 1C) as sole nitrogen source. The results show that the mutant plants exhibited slower growth in the medium contained xanthine or hypoxanthine compared to wild-type (Fig. 1C, lowest insert). The suboptimal growth of wild type lines is likely due to the low solubility of hypoxanthine and xanthine. In contrast, the growth on urea was the same for wild-type and XDH1 mutant transgenic plants (Fig. 1C). These results suggest that the conversion of xanthine to metabolically active intermediates, such as ureides and urea synthesized through XDH1, can play a role in ensuring nutrient supply for normal plant growth in purine containing media. Indeed, urea has been shown to be essential for the germination of Arabidopsis under nitrogenlimited conditions,¹² and recent studies have also shown that uric acid,¹³ allantoin and allantoate,^14–16 can serve as the sole nitrogen source during the growth of Arabidopsis plants. Taken together, the data suggest that ureide formation is an active component of normal plant metabolism facilitating the recovery of nitrogen in stress and non-stressed metabolism in a manner analogous to legumes. Indeed, legumes arose about 50–55 milion years ago¹⁷ and likely recruited and amplified existing plant functional purine pathways for their efficient nitrogen distribution system.     

Genetic and metabolic regulation of purine base transport in Neurospora crassa.

Summary Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. The early purine catabolic enzymes in this organism are regulated by induction and by ammonium repression. Studies were undertaken to investigate purine base transport and its regulation in Neurospora. The results of competition experiments with uric acid and xanthine transport strongly suggest that uric acid and xanthine share a common transport system. It was also shown that the common transport system for uric acid and xanthine is distinct from a second transport system shared by hypoxanthine, adenine and guanine, and apparently also distinct from the transport system(s) for adenosine, cytosine and uracil. Regulation of the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system was studied. The results reveal that the uric acid-xanthine transport system is regulated by ammonium repression, but does not require uric acid induction. Neither ammonium repression nor uric acid induction controls the hypoxanthine-adenine-guanine transport system. A gene, designated amr, which is believed to be a positive regulatory gene for nitrogen metabolism of Neurospora crassa, was found to dramatically affect both the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system. A model for the action of the amr locus as a positive regulatory gene and for the interaction between the amr gene product and its recognition sites will be discussed.     

Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD⁺ also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD⁺-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD⁺-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD⁺ by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD⁺ synthesis were overexpressed and led to a 221% increase in the NAD⁺ concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD⁺ content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD⁺ by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD⁺ was constructed, with the intracellular NAD⁺ concentration reaching 26.9 μmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD⁺-producing strain through global regulation metabolic engineering.     

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility

Nicotinamide adenine dinucleotide (NAD⁺) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD⁺ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD⁺ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD⁺ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD⁺ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD⁺ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD⁺ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.     

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD⁺. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD⁺, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD⁺ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD⁺ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD⁺ precursors, or a combination of KP inhibitors with NAD⁺ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD⁺ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD⁺ metabolism can synergistically increase life span.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.