Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices.

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5'-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-(14)C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.     

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.     

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.)

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O₂ treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O₂-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N₂ supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.     

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently inEscherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved inde novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed inE. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced inE. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.     

De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. coli.

The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E. coli mutant using an avian liver cDNA expression library. In E. coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins. The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities. Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced. The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins. Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS. The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis. Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.     

The apo and ternary complex structures of a chemotherapeutic target: human glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.     

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.     

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.

     

GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis

Glycinamide ribonucleotide formyltransferase (GART) has been established as a pivotal enzyme in de novo purine synthesis, and mediates cellular apoptosis in many diseases. We aimed to investigate the role of GART in the pathogenesis of Crohn’s disease (CD). In our study, we demonstrated for the first time that GART expression is up-regulated in patients with active CD and in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model. Moreover, the inhibition of GART induced cellular apoptosis and suppressed the migration of IECs through the activation of the MEKK3-MKK3-p38 mitogen-activated protein kinase (MAPK) pathway, following with the dys-regulation of p53 and p53 up-regulated modulator of apoptosis (PUMA). Taken together, GART plays a critical role in the protection of cellular apoptosis and migration of intestinal epithelial cells to maintain the integrity of the epithelial barrier, thus providing a new potential approach in designing a novel therapy for CD.     

Uricase from soybean root nodules: Purification,properties, and comparison with the enzyme from cowpea

A 45-fold purification of uricase (urate:O₂ oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 ± 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed K_m values of 10 and 31 μm for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (K_i = 10 μm). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of M_r 50,000 at pH 7.5.     

Identification of potential inhibitors for AIRS from de novo purine biosynthesis pathway through molecular modeling studies – a computational approach

In cancer, de novo pathway plays an important role in cell proliferation by supplying huge demand of purine nucleotides. Aminoimidazole ribonucleotide synthetase (AIRS) catalyzes the fifth step of de novo purine biosynthesis facilitating in the conversion of formylglycinamidine ribonucleotide to aminoimidazole ribonucleotide. Hence, inhibiting AIRS is crucial due to its involvement in the regulation of uncontrollable cancer cell proliferation. In this study, the three-dimensional structure of AIRS from P. horikoshii OT3 was constructed based on the crystal structure from E. coli and the modeled protein is verified for stability using molecular dynamics for a time frame of 100 ns. Virtual screening and induced fit docking were performed to identify the best antagonists based on their binding mode and affinity. Through mutational studies, the residues necessary for catalytic activity of AIRS were identified and among which the following residues Lys35, Asp103, Glu137, and Thr138 are important in determination of AIRS function. The mutational studies help to understand the structural and energetic characteristics of the specified residues. In addition to Molecular Dynamics, ADME properties, binding free-energy, and density functional theory calculations of the compounds were carried out to find the best lead molecule. Based on these analyses, the compound from the NCI database, NCI_121957 was adjudged as the best molecule and could be suggested as the suitable inhibitor of AIRS. In future studies, experimental validation of these ligands as AIRS inhibitors will be carried out.     

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES

The effect of O₂ and pH on the in vitro synthesis of ¹⁴C-labeled ureides from [8-¹⁴C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O₂ concentrations and low pH, ureide synthesis was inhibited and the ¹⁴C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O₂ concentrations and high pH, also stimulated ureide synthesis, and the ¹⁴C label was incorporated principally into allantoin. The overall response of the system to O₂ concentration and pH indicated that the per cent distribution of total ¹⁴C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-¹⁴C]hypoxanthine was also inhibited.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.     

Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.