The biochemistry of nitrogen mobilization: purine ring catabolism

The enzymatic route of purine ring catabolism has recently been completed by the discovery of several novel enzymes identified through comparative genome analyses. Here, we review these recent discoveries and present an overview of purine ring catabolism in plants. Xanthine is oxidized to urate in the cytosol, followed by three enzymatic steps taking place in the peroxisome and four reactions in the endoplasmic reticulum releasing the four ring nitrogen as ammonia. Although the main physiological function of purine degradation might lie in the remobilization of nitrogen resources, it has also emerged that catabolic intermediates, the ureides allantoin and allantoate, are likely to be involved in protecting plants against abiotic stress. Conserved alternative splicing mediating the peroxisomal as well as cytosolic localization of allantoin synthase potentially links purine ring catabolism to brassinosteroid signaling.     

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.     

Update on ureide degradation in legumes

Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two 'additional' enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N2-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N2-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

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.     

Is xanthine dehydrogenase involved in response of pea plants (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) to salinity or ammonium treatment?

The effect of salinity and different nitrogen sources on the level of xanthine dehydrogenase (XDH) activity in roots and leaves of pea plants was investigated. Two bands of xanthine dehydrogenase activity (XDH-R2, XDH-R3) were detected in roots after native PAGE and staining with hypoxanthine as substrate. Only one band of XDH activity (XDH-L1) was detected in leaf extracts. Within leaves of three different ages the highest XDH activity was detected in young leaves both under control as well as stress conditions. Salinity did not affect significantly the activity of XDH in pea roots, however, depressed XDH activity in leaves. A significant increase of XDH activity both in roots and leaves was observed only when ammonium was applied as the sole N source. Increased concentration of ureides in the xylem sap of pea plants was observed for both ammonium and high salt treatments, although the higher content of ureides in the xylem sap of 100 mM NaCl treated plants may be rather a result of lower rate of exudation from roots than of increased root ureide biosynthesis. Thus, the changes of root and leaf XDH activity in pea plants seem to be tightly correlated with ureide synthesis that is induced by NH ₄ ⁺ , the product of N fixation, and rather than by salinity. A contribution of pea XDH in increased oxygen species or uric acid production under saline conditions seems to be less than likely.     

Enzymes of Purine Biosynthesis and Catabolism in Glycine max: I. COMPARISON OF ACTIVITIES WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING NODULE DEVELOPMENT

During the period examined from 12 to 63 days after planting, the ureides, allantoin and allantoic acid, were the predominant nitrogenous solutes in the xylem exudate of soybeans (Glycine max [L.]) growing solely on symbiotically fixed nitrogen, accounting for approximately 60% and greater than 95% of the total nitrogen in the xylem exudate before and after the onset of active nitrogen fixation, respectively. For plants between 18 and 49 days of age, the apparent rate of ureide export estimated from concentrations of ureides in xylem exudate collected over a period of one hour was closely related to the rate of nitrogen fixation estimated from measurements of C₂H₂ reduction by nodulated root systems. After this time, the apparent rate of ureide export per plant continued to increase, reaching a maximum value at day 63 of 12 micromoles per plant per hour, even though the rate of C₂H₂ reduction per plant declined approximately four-fold. The most probable pathway for the biosynthesis of ureides involves the catabolism of purines. The levels of phosphoribosylpyrophosphate (PRPP) synthetase, which catalyzes the formation of the PRPP required for purine synthesis, increased in parallel with the rates of nitrogen fixation (C₂H₂) from day 18 reaching a maximum value of 13.9 micromoles per plant per hour at day 49, and then both activities declined rapidly. During the period of active nitrogen fixation the ratio of PRPP synthesis estimated from measurements of PRPP synthetase activity in cell-free extracts to the apparent rate of ureide export was between 1 and 2. The activities of the enzymes of purine catabolism, xanthine dehydrogenase, uricase, and allantoinase, increased in parallel with the increases in nodule mass and the export of ureides with maximum activities of 13, 119, and 79 micromoles per plant per hour, corresponding with apparent rates of ureide export in the range of 9.5 to 11.9 micromoles per plant per hour. These results demonstrate that there is a close association between nitrogen fixation, PRPP synthetase activity, and ureide export in soybeans and support the proposal that recently-fixed nitrogen is utilized in the de novo synthesis of purines which are subsequently catabolized to produce the ureides.     

Ureide metabolism in higher plants

The synthesis, transport and assimilation of the ureides, allantoin and allantoic acid, in higher plants is reviewed. Evidence indicates that in nodulated legumes ureides are synthesized from products of N₂-fixation via purine synthesis and degradation. Their synthesis in other plants also appears to be via purine degradation but is dependent on the inorganic nitrogen source fed to the plant; greatest ureide production is associated with ammonium assimilation. The use of ureides rather than amides for N-transport from the root to the shoot via the xylem stream results in an improved carbon economy of the plant. Good evidence for the transport of ureides in the phloem is lacking for most species examined although it is assumed to be important, particularly in fruit and seed development. Ureides are stored and assimilated mainly in the shoot. The precise pathways, localization and regulation of ureide assimilation are poorly understood and require further investigation. Similarities exist between the properties of the enzymes involved in ureide assimilation in higher plants and in micro-organisms. However, the evidence that light appears to be involved in ureide assimilation in green tissues suggests that different regulatory mechanisms may exist in plants compared with micro-organisms. The economically important legume crops such as soybeans, cowpeas and Phaseolus sp. are all ureide producers. To aid our understanding of the productivity of these plants knowledge of how ureide-N is converted into seed protein is essential.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

The RNAi-mediated silencing of xanthine dehydrogenase impairs growth and fertility and accelerates leaf senescence in transgenic Arabidopsis plants

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.     

Urease Is Not Essential for Ureide Degradation in Soybean

The hypothesis that soybean (Glycine max L. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-e1/eu3-e1) plants were supported by fixed N2 or by 2 mM NH4NO3, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3% the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [14C-ureido]allantoate to [14C]urea, whereas 40 to 70% of [14C-guanido]arginine was recovered as [14C]urea.     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Enzymes of ammonia assimilation in legume nodules: A comparison between ureide- and amide-transporting plants

Levels of amide and ureide biogenic enzymes were compared in the plant cytosol fractions of root nodules from soybean ( Glycine max L. Merr., cv. Williams), pintobean ( Phaseolus vulgaris L. cv. Pinto) and Lupin ( Lupinus angustifolius L. cv. Frost). Enzymes of purine oxidation were found to be present in significant quantities only in ureide-transporting pintobean and soybean nodules. The levels of these enzymes were low in lupin, but this amide-exporter had significantly higher levels of asparagine synthetase. Enzymes of de novo purine biosynthesis and glycine biosynthesis were present at higher levels in pintobean and soybean, consistent with a role for de novo purine biosynthesis in ureide biogenesis. The low levels of these enzymes in lupin are consistent with a role in general purine and amino acid metabolism in these nodules, not directly related to the synthesis of transport compounds for fixed atmospheric nitrogen. Amino acid concentrations in soybean, pintobean and lupin nodules reflected the metabolic differences between amide and ureide plants. The comparative data presented are consistent with a pathway of ureide biogenesis using glutamine, glutamate and aspartate synthesized via reactions catalyzed by glutamine synthetase, glutamate synthase and aspartate aminotransferase in the de novo synthesis of purines followed by oxidation of these purines to produce the ureides allantoin and allantoic acid.     

Effect of water stress on the enzymes of nitrogen metabolism in mung bean (Vigna radiata Wilczeck) nodules

Abstract. Water stress created by withholding irrigation in mung bean resulted in decreased leaf water potential and nodule moisture content. Decreased leaf water potential was associated with decreased activity of nitrogenase, glutamine synthetase (GS), asparagine synthetase (AS), aspartate amino transferase (AAT), xanthine dehydrogenase (XDH) and uricase. However, the activity of glutamate dehydrogenase increased three-fold under severe stress. The activity of allantoinase and allantoicase was not affected by moderate stress but decreased under severe stress. The in vitro production of allantoic acid from allantoin and uric acid in the cytosol fraction decreased more than its production from xanthine and hypoxanthine. The production of NADH also decreased under stress.
During recovery from severe stress, the activity of XDH and uricase further decreased, whilst that of allantoinase and allantoicase increased compared to the control. This corresponded with the higher content of ureides during recovery. The recovery in other enzymes was not complete although leaf water potential and nodule moisture content recovered fully within 24 h.     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.