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.

     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.

Labeling studies using ¹⁴CO₂ and ¹⁵N₂ and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.

Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.

Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.

     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen 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 identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

Metabolism and translocation of fixed nitrogen in the nodulated legume

Summary Nitrogen (N₂) fixed by Rhizobium bacteroids in the legume nodule is excreted as ammonia to the surrounding host cell where it is efficiently assimilated into the amide group of glutamine. Generally glutamine is a minor exported solute of nitrogen, being further metabolised to asparagine in temperate species and to the ureides, allantoin and allantoic acid in tropical species. These solutes serve as the principal translocated forms of nitrogen in xylem. Compartmentalisation of the pathways of nitrogen metabolism and the role of ammonia in regulation of their activity is examined in nodules of both asparagine-forming (Lupinus albus L.) and ureide-forming (Vigna unguiculata L. Walp) symbioses.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.     

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.     

Vascular transport and soybean nodule function: II. A role for phloem supply in product export*

Abstract The ureide content of soybean (Glycine max (L.) Merr.) nodules was unaffected by variations in the transpirational rate, while whole plant manipulations designed to decrease phloem supply to nodules resulted in lower rates of nitrogenase activity and an increase in the ureide content of the nodules. The rate of ureide export from the nodule was estimated from the exponential rate of decrease in the pool size of ureides in nodules, following exposure to an N₂-free atmosphere (Ar:O₂). Export was greatly reduced under treatments which reduced phloem supply to the nodule. A water budget for nodules suggested that the delivery of water to the nodule via mass flow in the phloem was comparable to that required for export of ureides from the nodule in the xylem from the nodule. Therefore, we suggest that xylem export from nodules is related to the phloem supply to the nodule rather than to the transpirational flux in the parent root. This suggestion is related to the reported decreases in nodule permeability to gases under conditions of phloem deprivation.     

Root nodule anatomy, type of export product and evolutionary origin in some Leguminosae

Abstract. The tribe Phaseoleae, of the sub-family Papi-lionoideae of the Leguminosae shows distinct differences from the tribes Vicieae and Trifolieae in nodule morphology and anatomy. Nodules of the Phaseoleae have determinate growth as, at maturity, the vascular strands fuse at the apex forming, effectively, a closed loop of the root stele. Nodules of the Vicieae and Trifolieae have an apical meristem, hence indeterminate growth; one or more branches of the root stele enter and dichotomise within the nodule, new elements are differentiated in relation to nodule growth, and the fine branches are free at the apical end of the nodule. Nodules of the Vicieae and Trifolieae additionally have vascular transfer cells and vacuolate infected cells, and the rhizobial bacteroids are pleomorphic.
The principal export products of nitrogen fixing nodules of the Phaseoleae are the ureides allantoin and allantoic acid, whilst those of the Vicieae and Trifolieae are amides and amino acids, especially glutamine and asparagine. The advantages and disadvantages of these export products are discussed in the light of nodular vascular anatomy and in respect of the tropical/subtropical origin of the Phaseoleae and the temperate origin of the Vicieae and Trifolieae.     

Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes

Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to ¹⁴CO₂ to investigate the contribution of nodule CO₂ fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse-chase experiments indicated that CO₂ fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO₂ fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO₂ fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.     

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     

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.     

Re-evaluation of the role of ureides in the xylem transport of nitrogen in Arachis species

There are conflicting reports in the literature of the possible role of the ureides, allantoin and allantoic acid, in the nitrogen economy of Arachis species. Therefore, xylem sap composition in food peanut ( Arachis hypogaea L.), and two forage peanuts ( A. pintoi L. and A. glabrata Benth.) has been studied in detail. Xylem saps were collected from peanuts grown under different nutritional regimes and environmental conditions in the glasshouse and field in Australia, Malaysia and Indonesia, and the N-containing solutes analysed. The relative amounts and concentrations of ureides in these peanut exudates were compared with those of soybean ( Glycine max [L.] Merr.) – a species known to export ureides in its xylem stream as the major product of N₂ fixation.
Xylem concentrations of ureides in soybean were high in N₂-fixing plants (2.9 to 3.7 μmol ml⁻¹), representing 60 to 88% of xylem solute nitrogen, but it contributed only 9% (0.7 μmol ml⁻¹) if plants were unnodulated and supplied nitrate. In all species of peanut, concentrations of ureides measured in xylem sap were generally much smaller (0.02 to 0.37 μmol ml⁻¹; 1–7% of xylem nitrogen) and were unaffected by peanut species or cultivar, rhizobial strain, plant size, growth rate, or stage of development, and were not related to N₂ fixation (less than 0.1% of currently fixed nitrogen exported as ureides) or the assimilation of nitrate. Apparently high levels of ureides in sap from some field-grown plants were shown to be due to interference with the ureide colorimetric assay by some contaminating compound rather than represent increased ureides per se.     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

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.     

Role of amides, amino acids, and ureides in the nutrition of developing soybean seeds

The various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos.     

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.     

5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: kinetic and regulatory properties

Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de novo purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent Mr of 8 X 10(6), but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required Mg2+, and PRPPMg3- was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the Vm with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine, PPi, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.