Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions.

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non-leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1OX ) under several N regimes. Under normal field conditions, panicles from OsUPS1OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N-starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low-N soil supplemented with different N-concentrations, OsUPS1OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole-body-overexpressing plants driven by the GOS2 promoter (OsUPS1GOS2 ) as well as silencing plants (OsUPS1RNAi ). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1GOS2 while in OsUPS1RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.

AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes.

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production.

Allantoin catabolism influences the production of antibiotics in Streptomyces coelicolor.

Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.

Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae.

Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in purine bases. In Klebsiella pneumoniae, the genes responsible for the assimilation of purine ring nitrogen are distributed in three separated clusters. We characterized the gene cluster involved in the metabolism of allantoate (genes KPN_01761 to KPN_01771). The functional assignments of HpxK, as an allantoate amidohydrolase, and of HpxU, as a regulator involved in the control of allantoate metabolism, were assessed experimentally. Gene hpxU encodes a repressor of the RpiR family that mediates the regulation of this system by allantoate. In this study, the binding of HpxU to the hpxF promoter and to the hpxU-hpxW intergenic region containing the divergent promoter for these genes was evidenced by electrophoretic mobility shift assays. Allantoate released the HpxU repressor from its target operators whereas other purine intermediate metabolites, such as allantoin and oxamate, failed to induce complex dissociation. Sequence alignment of the four HpxU identified operators identified TGAA-N8-TTCA as the consensus motif recognized by the HpxU repressor.

Urate as a physiological substrate for myeloperoxidase: implications for hyperuricemia and inflammation.

Urate and myeloperoxidase (MPO) are associated with adverse outcomes in cardiovascular disease. In this study, we assessed whether urate is a likely physiological substrate for MPO and if the products of their interaction have the potential to exacerbate inflammation. Urate was readily oxidized by MPO and hydrogen peroxide to 5-hydroxyisourate, which decayed to predominantly allantoin. The redox intermediates of MPO were reduced by urate with rate constants of 4.6 × 10(5) M(-1) s(-1) for compound I and 1.7 × 10(4) M(-1) s(-1) for compound II. Urate competed with chloride for oxidation by MPO and at hyperuricemic levels is expected to be a substantive substrate for the enzyme. Oxidation of urate promoted super-stoichiometric consumption of glutathione, which indicates that it is converted to a free radical intermediate. In combination with superoxide and hydrogen peroxide, MPO oxidized urate to a reactive hydroperoxide. This would form by addition of superoxide to the urate radical. Urate also enhanced MPO-dependent consumption of nitric oxide. In human plasma, stimulated neutrophils produced allantoin in a reaction dependent on the NADPH oxidase, MPO and superoxide. We propose that urate is a physiological substrate for MPO that is oxidized to the urate radical. The reactions of this radical with superoxide and nitric oxide provide a plausible link between urate and MPO in cardiovascular disease.

Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant.

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID]=0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID=0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.

Conserved alternative splicing of Arabidopsis transthyretin-like determines protein localization and S-allantoin synthesis in peroxisomes.

S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL(1-)) and the absence (TTL(2-)) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL(1-) localizes in peroxisomes, whereas TTL(2-) localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.

Intrinsic reactivity of uric acid with dioxygen: Towards the elucidation of the catalytic mechanism of urate oxidase.

Urate oxidase catalyzes the transformation of uric acid in 5-hydroxyisourate, an unstable compound which is latter decomposed into allantoïn. Crystallographic data have shown that urate oxidase binds a dianion urate species deprotonated in N3 and N7, while kinetics experiments have highlighted the existence of several intermediates during catalysis. We have employed a quantum mechanical approach to analyze why urate oxidase is selective for one particular dianion and to explore all possible reaction pathways for the oxidation of one uric acid species by molecular dioxygen in presence of water. Our results indicate the urate dianion deprotonated in N3 and N7 is among all urate species that can coexist in solution it is the compound which will lose the most easiestly one electron in presence of molecular dioxygen. In addition, the transformation of this dianion in 5-hydroxyisourate is thermodynamically the most favorable reaction. Finally, several reaction pathways can be drawn, each starting with the spontaneous transfer of one electron from the urate dianion to molecular dioxygen. During that period, the existence of a 5-hydroperoxyisourate intermediate, which has been proposed elsewhere, does not seem mandatory.

Presence of epidermal allantoin further supports oxidative stress in vitiligo.

Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H(2)O(2) yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H(2)O(2) up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H(2)O(2) in a concentration-dependent manner, where concentrations of 10(-6 )m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H(2)O(2)-mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10(-3 )m H(2)O(2). Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H(2)O(2)-mediated oxidative stress in vitiligo via H(2)O(2)-production and allantoin formation in the epidermal compartment.

Ureide biosynthesis in legume nodules.

In tropical legumes like Glycine, Phaseolus and Vigna sp., ammonia as direct product of symbiotic nitrogen fixation is converted to ureides (allantoin and allantoic acid) and they were translocated to the shoots as nitrogen source. In the xylem sap of soybean in reproductive phase the ureides reached to 60-75% of soluble nitrogen. In nodules infected cells (plastid and mitochondria) and uninfected cells (peroxisome) shares de novo purine biosynthesis and urate oxidation to produce ureides respectively. Current research revealed unique feathers on this symbiotic metabolism, especially on regulation of purine biosynthesis, uricase gene expression and feedback inhibition of ureides to nitrogen fixing activity.

Functional expression and peroxisomal targeting of rat urate oxidase in monkey kidney cells.

Humans and hominoid primates lack the enzyme urate oxidase, which catalyzes the oxidation of uric acid to allantoin. In rats and most other mammals, urate oxidase is present as a crystalloid core within the peroxisomes of liver parenchymal cells. To determine whether functionally active recombinantly expressed urate oxidase can be targeted to the peroxisome as well as display the crystalloid core-like structure, we expressed rat urate oxidase cDNA in African green monkey kidney cells (CV-1 cells) under the control of a cytomegalovirus promoter. Cell lines stably expressing urate oxidase were isolated. Northern blot analysis revealed a 1.3-kb transcript and immunoblot analysis confirmed the presence of urate oxidase in the stably transfected cells. The recombinant urate oxidase expressed in CV-1 cells was functionally active. Immunofluorescence microscopy revealed that the expressed protein was visualized as discrete granules in the cytoplasm. Electron microscopy and immunocytochemical localization studies showed that the recombinantly expressed protein formed distinct crystalloid core structures with bundles of tubules within single membrane limited cytoplasmic organelles. On cross section, the recombinant urate oxidase tubular structures are arranged as circles of 10 surrounding a slightly larger circle. This arrangement is reminiscent of urate oxidase-containing cores in rat liver peroxisomes. Immunocytochemical studies confirmed that the recombinantly expressed urate oxidase is correctly targeted to the catalase-containing peroxisomes in these CV-1 cells.

The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae.

We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

The role of nonparenchymal and parenchymal liver cells in the catabolism of extracellular purines.

Adenosine-degrading enzymes within the liver lobule can modulate both vascular and metabolic effects of circulating adenosine in the liver. Since it has not been fully established whether nonparenchymal cells participate in the elimination of sinusoidal purines, isolated Kupffer cells and endothelial cells were tested for their capacity to degrade extracellular purines. After perfusion and digestion of rat livers by collagenase, the resulting mixed cell population was separated by centrifugal elutriation. The isolated parenchymal and nonparenchymal cells were incubated for up to 2 hr in the presence of [8(-14)C]adenosine, [8(-14)C]guanosine and [8(-14)C]hypoxanthine (50 mumoles per liter). In the deproteinized medium, adenosine, guanosine, inosine, adenine, guanine, xanthine, hypoxanthine, uric acid and allantoin were separated by reversed-phase high-performance liquid chromatography. Radioactive peaks were collected and counted. Nonparenchymal cells catalyzed the degradation of adenosine into inosine and hypoxanthine. However, the formation of xanthine, uric acid or allantoin from adenosine could only be detected in hepatocyte suspensions. Within 15 min, adenosine was completely eliminated from the medium by Kupffer cells, whereas endothelial cells catabolized only less than half of the initial amount of the adenine nucleoside during this time period. Accordingly, incubation of nonparenchymal cells in the presence of hypoxanthine did not result in the formation of further breakdown products of the purine, whereas its catabolites slowly accumulated in the medium of hepatocytes. Guanosine conversion into guanine and xanthine was much slower in endothelial cells as compared to Kupffer cells and hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Salvage of circulating hypoxanthine by tissues of the mouse.

Hypoxanthine is an inefficient precursor of purine nucleotides in mouse tissues. In vitro, mouse erythrocytes salvage less than 10% of hypoxanthine (10 microM) added to whole blood in 30 min of incubation at 37 degrees C. In vivo, circulating hypoxanthine is rapidly degraded (greater than 90% in 10 min) to allantoin and uric acid. All tissues examined (other than erythrocytes) converted small amounts of hypoxanthine to nucleotides, with kidney and lung being the most active tissues examined. It is estimated that less than 2% of circulating hypoxanthine is salvaged in the mouse; the remainder is catabolized.

Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp.).

Fractionation of cell organelles of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp) by discontinuous and continuous sucrose density centrifugation indicated that starch-containing plastids possessed the complete pathway for purine nucleotide synthesis together with significant activities of some other enzymes associated with the provision of substrates in purine synthesis; triosephosphate isomerase (EC 5.3.1.1), NADH-glutamate synthase (EC 2.6.1.53), aspartate aminotransferase (EC 2.6.1.1), phosphoglycerate oxidoreductase (EC 1.1.1.95), and methylene tetrahydrofolate oxidoreductase (EC 1.5.1.5). Enzymes of purine oxidation, xanthine oxidoreductase (EC 1.2.3.2), and urate oxidase (EC 1.7.3.3) were recovered in the soluble fraction; glutamine synthetase (EC 6.3.1.2) occurred in bacteroids and in the cytosol. Intact, infected (bacteroid-containing) and uninfected cells were prepared by enzymatic maceration of the central zone of the nodule and partially separated by centrifugation on discontinuous sucrose gradients. Glutamine synthetase was largely restricted to infected cells whereas plastid enzymes, de novo purine synthesis, and urate oxidase were present in both cell types. Although the levels of all enzymes assayed were higher in infected cells, both cell types possessed the necessary enzyme complement for ureide formation. A model for the cellular and subcellular organization of nitrogen metabolism and the transport of nitrogenous solutes in cowpea nodules is proposed.

Purine catabolism in isolated rat hepatocytes. Influence of coformycin.

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [(14)C]adenine. The production of allantoin reached 32+/-5nmol/min per g of cells (mean+/-s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1mum-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50mum-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1mum-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50mum-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [(14)C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1mum-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50mum-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5'-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5'-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.