Discovery of a 3′,5′-guanosine monophosphate simulating factor in amoebae of dictyostelium discoideum

An enzyme degradable substance in an amoebae extract from Dictyostelium discoideum has been observed to exhibit properties of 3′,5′-guanosine monophosphate (cyclic GMP). This is the simplest differentiating system yet shown to contain cyclic GMP.     

Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

A direct, stimulating effect of cyclic GMP on purified phosphoribosyl pyrophosphate synthetase and its antagonism by cyclic AMP

The activity of phosphoribosyl pyrophosphate synthetase, purified from a line of rat hepatoma cells in continuous culture, is maximally stimulated (2–4 fold) by less than 10^?7M cyclic GMP. Half maximal stimulation occurs at 2 × 10^?9M. Cyclic GMP stimulates phosphoribosyl pyrophosphate synthetase by decreasing the Km of the enzyme for ATP from 50 μM to 10 μM without affecting the Vmax; it has no effect on the Km for ribose 5-phosphate, the other substrate. Cyclic AMP alone has no effect on the enzyme activity, but at micromolar concentrations it antagonizes the stimulation by cyclic GMP. GMP, GDP, and GTP do not stimulate enzyme activity; and AMP and ADP at micromolar concentrations do not antagonize the effect of cyclic GMP.There is no detectable cyclic nucleotide-activated protein kinase in the enzyme preparation. Cyclic GMP significantly stabilizes the enzyme to heat inactivation. We conclude that cyclic GMP binds directly to the enzyme in an allosteric fashion, causing it to have an increased affinity for one of its substrates, and that cyclic AMP directly antagonizes this effect.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

A developmentally regulated cysteine proteinase in Dictyostelium discoideum.

We have determined the sequence of a Dictyostelium mRNA encoding a protein with a high degree of homology to plant and animal cysteine proteinases. The degree of homology is highest in the region of the cysteine residue which is transiently acylated during peptide hydrolysis but all other residues known to be important in catalysis are also conserved. We have named this protein cysteine proteinase 1. There is a hydrophobic signal peptide of 18 amino acids and an additional 99 amino acids at the N terminus, which are not present in other cysteine proteases and which may be cleaved off during processing of the enzyme. There is a single copy of the gene in the Dictyostelium genome. The cysteine proteinase 1 mRNA is absent from growing cells and from cells isolated during the first 6 h of development but it constitutes approximately 1% of cellular mRNA by 10-12 h of development. During the development of Dictyostelium a major fraction of cellular protein is degraded to provide amino acids and a source of energy. Cysteine proteinase 1 may play a role in this auto-digestion.     

Aminoacyl-tRNA-synthetases and their high molecular weight complexes in the regenerating rat liver

Glutamyl- and lysyl-tRNA synthetase activities in total preparations and high-molecular complexes from rat liver increase 21 h after partial hepatectomy. Glutamyl-tRNA synthetase has been highly purified from normal and regenerating liver. Km, Vmax values and molecular activity were practically the same for both enzyme preparations. Total methyltransferase activity and that as a part of high-molecular complexes increase 1.5 fold 21 h after the operation but the changes of individual methyltransferase activities differ. The level of tRNA methylation in vivo is also higher during regeneration. Proteinkinases of high-molecular complexes from regenerating liver are sensitive to cyclic AMP and GMP in contrast to control.     

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.     

Substrate Specificity and Oligomerization of Human GMP Synthetase

Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.     

Localization of glycogen synthetase during differentiation of presumptive cell types in Dictyostelium discoideum.

Ultramicrochemical techniques were utilized to assay glycogen synthetase (EC 2.4.1.11) activity in cell samples of Dictyostelium discoideum as small as 0.01 mug (dry weight) in reaction volumes of 0.1 mul. The activity was assayed by an amplification procedure employing the enzymatic cycling of pyridine nucleotides. These techniques were used to determine the extent of localization of glycogen synthetase in the two cell types during differentiation of D. discoideum. Localization studies in developing spore cells revealed decreasing enzyme activity to the culmination stage. During this phase of development, the enzyme required the presence of soluble glycogen for activity. From culmination to sorocarp stage, enzyme activity increased and was independent of the soluble glycogen. In developing stalk cells, synthetase showed a decreasing gradient of activity. In sorocarps, the cells in the stalk apex showed synthetase activity similar to that of the spores. The cells at the bottom of the stalk had no detectable activity.     

Purification and characterization of GMP synthetase from Yoshida sarcoma ascites cells

GMP synthetase was found in the cytosolic fraction of Yoshida sarcoma ascites cells. However, prolonged centrifugation resulted in precipitation of the enzyme. On sucrose density gradient centrifugation of a crude extract of Yoshida sarcoma ascites cells, a part of this enzyme showed high sedimentability at low ionic strength. On the basis of these observations, GMP synthetase was purified from Yoshida sarcoma ascites cells by means of procedures including centrifugal fractionation. The purified enzyme was shown to be homogeneous on SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel. The molecular weight of the GMP synthetase was estimated to be 78,000 by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-150. Its isoelectric point was estimated to be 5.6. The Km values of this enzyme for XMP, ATP, and glutamine were calculated to be 4.6, 120, and 300 microM, respectively. Although ammonia could substitute for glutamine as a donor of the amino group, the Km value was as high as 120 mM, indicating that it cannot be considered to be a physiological substrate. This enzyme showed high activity only in the presence of Mg2+, and very low activity in the presence of other divalent cations. Inhibition by nucleoside monophosphates was not significant. The enzyme required reduced sulfhydryl compounds for its activity.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

DdPDE4, a novel cAMP-specific phosphodiesterase at the surface of dictyostelium cells

Dictyostelium discoideum cells possess multiple cyclic nucleotide phosphodiesterases that belong either to class I enzymes that are present in all eukaryotes or to the rare beta-lactamase class II. We describe here the identification and characterization of DdPDE4, the third class I enzyme of Dictyostelium. The deduced amino acid sequence predicts that DdPDE4 has a leader sequence, two transmembrane segments, and an extracellular catalytic domain that exhibits a high degree of homology with human cAMP-specific PDE8. Expression of the catalytic domain of DdPDE4 shows that the enzyme is a cAMP-specific phosphodiesterase with a K(m) of 10 microm; cGMP is hydrolyzed at least 100-fold more slowly. The full-length protein is shown to be membrane-bound with catalytic activity exposed to the extracellular medium. Northern blots and activity measurements reveal that expression of DdPDE4 is low during single cell stages and increases at 9 h of starvation, corresponding with mound stage. A function during multicellular development is confirmed by the phenotype of ddpde4(-) knock-out strains, showing normal aggregation but impaired development from the mound stage on. These results demonstrate that DdPDE4 is a unique membrane-bound phosphodiesterase with an extracellular catalytic domain regulating intercellular cAMP during multicellular development.     

The Selenophosphate synthetase gene from Leishmania major.

Selenophosphate synthetase coding sequence was cloned from Leishmania major by RT-PCR amplification. The DNA sequence was found to have an open reading frame encoding protein with 398 amino acids and does not have in-frame UGA codon. The deduced amino acid sequence indicates that it has cysteine residue instead of selenocysteine at the active site of enzyme. Amino acid sequence alignment of Selenophosphate synthetase from parasite with the human enzyme showed approximately 45% homology. The sequences also indicated presence of conserved amino acid residues and motifs that are present in mammalian Selenophosphate synthetase. Southern analysis done with restriction enzyme digested genomic DNA and pulse filed separated chromosome suggests that L. major genome contain a single copy of Selenophosphate synthetase sequence. Expression analysis by Northern analysis and RT-PCR indicated Selenophosphate synthetase mRNA is present in promastigote and amastigote stages of parasite.     

Dual inhibitory effect of bredinin

Bredinin inhibition of cell growth was investigated in the mouse lymphoma cell line L5178Y. Bredinin caused the accumulation of IMP and the reduction of XMP. It was converted to the 5'-phosphate within the cells. Bredinin 5'-phosphate but not bredinin competitively inhibited both IMP dehydrogenase and GMP synthetase. Thus the inhibition of cell growth is probably due to bredinin 5'-phosphate, which inhibits the consecutive enzyme reactions IMP dehydrogenase and GMP synthetase. These inhibitions result in the accumulation of IMP and the reduction of XMP.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.

NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction. The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.     

Ammonia channeling in Plasmodium falciparum GMP synthetase: investigation by NMR spectroscopy and biochemical assays

GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with 15N-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium, (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.     

Inositol 1,4,5-trisphosphate induces cyclic GMP formation in Dictyostelium discoideum

Chemotactic signalling in the cellular slime mould Dictyostelium discoideum employs signalling molecules such as folate and cyclic AMP. These bind to specific cell surface receptors and rapidly trigger internal responses that induce chemotactic movement of the amoebae. Previous studies have shown that actin is polymerised within 3-5 sec of cyclic AMP or folate binding and that a peak of cyclic GMP is formed within 9-12 sec. Release of Ca2+ from intracellular stores has been implicated as a secondary messenger. Here we present evidence that D-myo-inositol 1,4,5-trisphosphate, when added to permeabilized amoebae of Dictyostelium, can mimic the action of chemoattractants on normal intact amoebae in inducing cyclic GMP formation. Our data suggest that IP3, which is known to act as an intermediary messenger between cell surface hormone receptors and release of Ca2+ from internal stores in mammalian cells, functions in a similar capacity during chemotaxis of this primitive eukaryote.     

Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum

Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.