The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.     

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

Background

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive.

Results

In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required.

Conclusions

Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.

     

Nucleic acid synthesis and nucleotide pools in purine-deficient Escherichia coli

1. The synthesis of nucleic acids and the content of purine nucleotides have been studied in selected purine-requiring strains of Escherichia coli including a purB(-) strain and a purB(-)guaA(-) strain. 2. When the exogenous purines can be converted into GTP but not into ATP, RNA is synthesized at the expense of intracellular ATP, ADP and AMP. 3. Net synthesis of RNA as measured by the incorporation of uracil can be correlated with the availability of GTP except when ATP falls to a very low concentration. 4. Nicotinamide nucleotides are not an important reservoir of adenine nucleotides for RNA synthesis.     

Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.     

Systematic variations in the content of the purine nucleotides in the steady-state perfused rat heart. Evidence for the existence of controlled storage and release of adenine nucleotides

1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1μg/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3μmol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem. 250, 6304–6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.     

Mechanism of adenine toxicity in Escherichia coli

The mechanism of adenine toxicity in an hpt gpt strain of Escherichia coli that is extremely sensitive to adenine inhibition was investigated. Adenine-resistant derivatives had secondary mutations in adeninephosphoribosyltransferase or the purR repressor. Growth studies with various purine salvage pathway mutants and the ability of guanosine to prevent adenine toxicity indicated that adenine exerts its toxic effects by depleting guanine nucleotide pools. In the presence of adenine, ATP pools increased twofold in wild-type cells and stabilized after 5 min. In contrast, ATP pools continued to rise in hpt gpt cells up to 25 min and increased sevenfold after adenine addition. hpt gpt cells were shown to have higher levels of adeninephosphoribosyltransferase than did the wild-type cells. In response to adenine addition, GTP pools dropped three- to fourfold in all strains tested. Although GTP levels returned to near normal values in wild-type cells after 35 min, no restoration of GTP pools was observed in the hpt gpt strain during this period. Measurements of guanine pools before and after the addition of adenine indicated that guaninephosphoribosyltransferase plays an important role in maintaining GTP pools by converting the free guanine to GMP during guanine nucleotide depletion.     

Molecular cloning and characterization of a cDNA encoding soybean nodule IMP dehydrogenase

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo purine biosynthesis and is a postulated key enzyme in nitrogen assimilation in ureide-exporting nodules. A 2016 bp cDNA for IMPDH, designated as IMPDH, was cloned from a soybean nodule cDNA library. IMPDH encodes a polypeptide of 502 amino acids with a predicted molecular weight of 53000 and a pI of 5.54. The deduced IMPDH is 70.5% identical to that in Arabidopsis, with a 100% homology in the putative active-site region. Expressing the cloned cDNA in Escherichia coli mutant strain KLC381 (DeltaguaB) restored IMPDH activity, permitting bacterial growth on minimal medium. Southern blot analysis suggested a single copy of IMPDH gene in the soybean genome. Northern blot analysis showed that the expression of IMPDH gene is apparently nodule-specific.     

Effect of lowered intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion.

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and intercoversion were studied using intact Ehrlich ascites tumor cells. The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.     

Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity

We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p < 0.05) and blocked adipogenesis (p < 0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p = 0.002) and adipocyte size (p = 0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass.     

Kinetics of Phosphate Incorporation into Adenosine Triphosphate and Guanosine Triphosphate in Bacteria

Escherichia coli cells were labeled with (33)PO(4) for several generations and then (32)PO(4) was added, samples were taken at various times, and adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were purified from an acid extract. The ratio of (32)P/(33)P in each of the six phosphate species was determined and compared to the ratio expected at isotopic steady state. The rate constants of the simplest network of pools which could explain the data, and were consistent with metabolic pathways, were then calculated. In the range of times studied (30 to 3,000 s at 21.5 C) the following generalizations can be made. (i) The gamma-phosphate at ATP requires 2,200 s to achieve 50% of its final specific activity and behaves as if it were exchanging with another species 4.4 times as large, with this other species turning over in 540 s. (ii) The beta-phosphate of ATP and the gamma-phosphate of GTP are in equilibrium with the gamma-phosphate of ATP. (iii) The specific activity of the gamma-phosphate of GTP lags 66 s behind the gamma-phosphate of ATP. (iv) The alpha-phosphates of ATP and GTP have equal specific activities which lag far behind the other four phosphates. A single precursor pool with a turnover time of 416s fits the data best, although a fair fit can be obtained assuming several sequential precursor pools with much shorter turnover times. These findings are consistent with known biosynthetic pathways and the probable flows through them, including the relationship of nucleotide biosynthesis to ribonucleic acid synthesis and turnover.     

Mononucleotide Metabolism in the Rat Brain After Transient Ischemia

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.     

Allosteric control of simian virus 40 T-antigen binding to viral origin DNA.

Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored.     

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced. Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 muM respectively, compared to 60 muM and 45 muM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib-PP synthetase activity was observed in both strains, although to a lesser extent in the mutant. Our data suggest that the mutant harbors a mutation in the structural gene for PRib-PP synthetase. The mutation responsible for the altered PRib-PP synthetase was located in the purB-hemA region at 26 min on the recalibrated linkage map.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.     

The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties

DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.