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Guanosine monophosphate (GMP) synthetase is an enzyme that converts xanthosine monophosphate to GMP. GMP plays an essential role in plant development and responses to internal and external stimuli. It also plays a crucial role in several plant physiochemical processes, such as stomata closure, cation flux regulation, pathogen responses and chloroplast development. The mRNA sequences of NtGMP synthase in tobacco (Nicotiana tabacum) were rapidly amplified from cDNA. The GMP synthase open reading frame contains a 1617 bp sequence encoding 538 amino acids. A sequence analysis showed that this sequence shares high homology with that of Nicotiana sylvestris, Nicotiana attenuata, N. tomentosiformis, Solanum tuberosum, Lycopersicon pennellii, L. esculentum, Capsicum annuum, C. chinense and C. baccatum GMP synthase. A BLAST analysis with a tobacco high-throughput genomic sequence database revealed that the tobacco GMP synthase gene has five introns and six exons. A phylogenetic analysis showed a close genetic evolutionary relationship with N. sylvestris GMP synthase. The tissue-specific expression profile was evaluated using quantitative real-time PCR. The data showed that NtGMP synthase was highly expressed in leaves and moderately expressed in roots, flowers, and stems. The subcellular localization was predicted using the WOLF PSORT webserver, which strongly suggested that it might be localized to the cytoplasm. In the current study, we cloned and comprehensively characterized GMP synthase in tobacco (Nicotiana tabacum). Our results establish a basis for further research to explore the precise role of this enzyme in tobacco.

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo., Dephosphorylation of xanthosine monophosphate (XMP) initiates purine nucleotide catabolism in plant cells. Here the authors identify an XMP phosphatase from Arabidopsis that channels XMP towards catabolism in vivo and demonstrate the structural basis for its XMP specificity.

In pediatric nephrotic syndrome, recommended mycophenolic acid (MPA) pharmacokinetics are higher than those for transplant recipients. In MPA therapeutic monitoring, inosine-5&prime, monophosphate dehydrogenase (IMPDH) activity may be useful. We modified the method established for renal transplant recipients and determined IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and children (4&ndash, 16 years) with nephrotic syndrome treated with mycophenolate mofetil (MMF). From children, four blood samples were collected, and MPA concentrations were also determined. IMPDH activity was calculated using xanthosine monophosphate (XMP) normalized with adenosine monophosphate (AMP), both determined with the HPLC-UV method. The modified method was accurate, precise, and linear for AMP and XMP within 0.50&ndash, 50.0 &mu, moL/L. Mean IMPDH activity in volunteers was 45.97 &plusmn, 6.24 &micro, moL&middot, s&ndash, 1&middot, moL&ndash, 1 AMP, whereas for children, the values were variable and amounted to 39.23 &plusmn, 27.40 &micro, 1 AMP and 17.97 &plusmn, 15.24 &micro, 1 AMP before the next MMF dose and 1 h afterward, respectively. The modified method may be applied to IMPDH activity determination in children with nephrotic syndrome treated with MMF. IMPDH activity should be determined after one thawing of PBMCs due to the change in AMP and XMP concentrations after subsequent thawing. For children, the lowest IMPDH activity was observed concomitantly with the highest MPA concentration.

The computational importance of guanosine monophosphate (GMP) comes from its importance in the synthesis of nucleic acids. Deamination reaction mechanisms, kinetics, and thermodynamics of GMP with 3H2O and 2H2O/OH− to produce xanthosine monophosphate (XMP) have been investigated. The proposed reactions were optimized in the gas-phase and in an aqueous medium. Two key steps were found for the proposed pathways: a tetrahedral intermediate formation and a single water-mediated six-membered ring transition state, yielding the desired product. The deamination of GMP with 2H2O/OH− yield deprotonated XMP− with only one pathway, which is highly exothermic, and the activation energy is significantly lower (33 kJ mol−1 at M11/6-31G(d)) compared to the other pathways. Our results suggest that pathway C is the most kinetically and thermodynamically favorable mechanism for the deamination of GMP with 3H2O with the lowest energy barrier of 123 kJ mol−1, due to multiple functions of water that enhance the reaction profile.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to xanthosine monophosphate, the rate-limiting step in de novo guanosine monophosphate (GMP) synthesis. In cultured cells, IMPDH polymerizes into micron-scale filamentous structures when GMP synthesis is inhibited by depletion of purine precursors or by various drugs, including mycophenolic acid, ribavirin, and methotrexate. IMPDH filaments also spontaneously form in undifferentiated mouse embryonic stem cells and induced pluripotent stem cells, hinting they might function in various highly proliferative cell types. Therefore, we investigated IMPDH filament formation in human and murine T cells, which rely heavily on de novo guanine nucleotide synthesis to rapidly proliferate in response to antigenic challenge. We discovered extensive in vivo IMPDH filament formation in mature T cells, B cells, and other proliferating splenocytes of normal, adult B6 mice. Both cortical and medullary thymocytes in young and old mice also showed considerable assembly of IMPDH filaments. We then stimulated primary human peripheral blood mononuclear cells ex vivo with T cell mitogens phytohemagglutinin (PHA), concanavalin A (ConA), or antibodies to CD3 and CD28 for 72 h. We detected IMPDH filaments in 40–60% of T cells after activation compared to 0–10% of unstimulated T cells. Staining of activated T cells for the proliferation marker Ki-67 also showed an association between IMPDH filament formation and proliferation. Additionally, we transferred ovalbumin-specific CD4+ T cells from B6.OT-II mice into B6.Ly5a recipient mice, challenged these mice with ovalbumin, and harvested spleens 6 days later. In these spleens, we identified abundant IMPDH filaments in transferred T cells by immunofluorescence, indicating that IMPDH also polymerizes during in vivo antigen-specific T cell activation. Overall, our data indicate that IMPDH filament formation is a novel aspect of T cell activation and proliferation, and that filaments might be useful morphological markers for T cell activation. The data also suggest that in vivo IMPDH filament formation could be occurring in a variety of proliferating cell types throughout the body. We propose that T cell activation will be a valuable model for future experiments probing the molecular mechanisms that drive IMPDH polymerization, as well as how IMPDH filament formation affects cell function.

Plants can fully catabolize purine nucleotides. A firmly established central intermediate is the purine base xanthine. In the current widely accepted model of plant purine nucleotide catabolism, xanthine can be generated in various ways involving either inosine and hypoxanthine or guanosine and xanthosine as intermediates. In a comprehensive mutant analysis involving single and multiple mutants of urate oxidase, xanthine dehydrogenase, nucleoside hydrolases, guanosine deaminase, and hypoxanthine guanine phosphoribosyltransferase, we demonstrate that purine nucleotide catabolism in Arabidopsis (Arabidopsis thaliana) mainly generates xanthosine, but not inosine and hypoxanthine, and that xanthosine is derived from guanosine deamination and a second source, likely xanthosine monophosphate dephosphorylation. Nucleoside hydrolase 1 (NSH1) is known to be essential for xanthosine hydrolysis, but the in vivo function of a second cytosolic nucleoside hydrolase, NSH2, is unclear. We demonstrate that NSH1 activates NSH2 in vitro and in vivo, forming a complex with almost two orders of magnitude higher catalytic efficiency for xanthosine hydrolysis than observed for NSH1 alone. Remarkably, an inactive NSH1 point mutant can activate NSH2 in vivo, fully preventing purine nucleoside accumulation in nsh1 background. Our data lead to an altered model of purine nucleotide catabolism that includes an NSH heterocomplex as a central component.

Summary The high‐affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance in the rice blast fungus Magnaporthe oryzae. To explore the underlying mechanism, we identified MoImd4 as an inosine‐5′‐monophosphate dehydrogenase (IMPDH) homologue that interacts with MoPdeH. Targeted deletion of MoIMD4 resulted in reduced de novo purine biosynthesis and growth, as well as attenuated pathogenicity, which were suppressed by exogenous xanthosine monophosphate (XMP). Treatment with mycophenolic acid (MPA), which specifically inhibits MoImd4 activity, resulted in reduced growth and virulence attenuation. Intriguingly, further analysis showed that MoImd4 promotes the phosphodiesterase activity of MoPdeH, thereby decreasing intracellular cAMP levels, and MoPdeH also promotes the IMPDH activity of MoImd4. Our studies revealed the presence of a novel crosstalk between cAMP regulation and purine biosynthesis in M. oryzae, and indicated that such a link is also important in the pathogenesis of M. oryzae.

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.

Guanosine monophosphate synthetase (GMPS) is a glutamine amidotransferase catalyzing amination of xanthosine monophosphate (XMP) to form GMP. It is a enzyme involved in de novo purine biosynthesis. T and B lymphocytes, and certain cancer cells, such as malignant melanoma are dependent on de novo synthesis of purine nucleotide for activation and proliferation. GMPS is an attractive target for drug discovery and development to treat autoimmune diseases, organ transplant rejection, and cancers. Mycophenolate acid (MPA), an active ingredient of Mycophenolate Mofetil (Cellcept) has been used for organ transplantation and autoimmune diseases. MPA is a natural product which inhibits inosine monophosphate dehydrogenase (IMPDH), an enzyme upstream of GMPS. Using IMTACTM (Isobaric Mass Tagged Affinity Characterization) platform to perform global proteome ligand screen in live cancer cells, a highly potent and highly selective irreversible inhibitor against GMPS was discovered. Here we report the identification, in vitro characterization and in vivo evaluation of this molecule BGS2019. Target engagement of BGS2019 was determined by In-Gel-Fluorescent assay in cell with an EC50 of 15.6nM. It inhibited GMPS enzymatic activity with an IC50 of 6nM. The mechanism of BGS2019 in cell-based assays was tested, which showed the accumulation of IMP (the substrate for GMPS) and the reduction of GMP, GDP and GTP, upon BGS2019 treatment. BGS2019 was evaluated for its cell growth inhibition activity in several cancer cell lines and it suppressed cancer cell growth with IC50 values from 30nM to 100nM. In vivo administration of BGS2019 was well tolerated in mice and exhibited tumor suppression in animal model. Citation Format: Hang Chen, Heather Ha, Robert Stanley, Cindy Huang, Qian Cai, Irene Yuan, Ping Cao. Discovery of BGS2019, a highly potent and selective covalent inhibitor of GMPS using IMTACTM platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B135. doi:10.1158/1535-7163.TARG-19-B135

Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2'-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in purine nucleotide biosynthesis. It is responsible for catalyzing the oxidation of inosine monophosphate (IMP) into xanthosine monophosphate (XMP). Concurrently, the cofactor NAD+ is reduced to NADH. Poly(ADP-ribose) polymerase 1 (PARP-1) also utilizes NAD+ as a substrate to synthesize poly(ADP-ribose). It has been demonstrated that inhibition of PARP-1 activity can be an effective cancer therapeutic. However, most PARP-1 inhibitors, including olaparib, were developed as NAD+ analogs. Therefore, these inhibitors likely interfere with other NAD+-dependent pathways such as the one involved in de novo purine metabolism. In this chapter, we describe a method to quantitatively measure IMPDH activity by taking advantage of the autofluorescence of the product NADH. We use this method to analyze the effects of olaparib and non-NAD+-like PARP-1 inhibitor (5F02) on IMPDH activity. We found that olaparib, unlike 5F02, significantly inhibits IMPDH activity in a dose-dependent manner. Our results suggest that IMPDH inhibition is an off-target effect of olaparib treatment. The consequences of this effect should be addressed by future clinical studies.

Combinations of immunosuppressive drugs are routinely used post-transplantation to prevent rejection and/or other complications and optimize outcomes. The prodrug ester mycophenolate mofetil (MMF) is frequently used in solid-organ and stem cell transplantation settings. A growing body of evidence supports therapeutic monitoring of this immunosuppressant to optimize its efficacy and reduce toxicity. Thus, pharmacodynamic monitoring of MMF is a strategy that could potentially improve patient outcomes. Pharmacodynamic measurements require evaluation of inosine-5'-monophosphate dehydrogenase (IMPDH) activity, the target enzyme of the active moiety mycophenolic acid. Various nonradioactive methods using chromatographic separations have been used to quantify xanthosine monophosphate, the catalytic product of the enzyme, to indirectly evaluate IMPDH activity. However, no methods have used mass spectrometry based detection, which provides more specificity and sensitivity. Here, we describe a liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method for the quantification of xanthosine monophosphate and adenosine monophosphate (for normalization) in lysates of peripheral blood mononuclear cells (PBMCs) from hematopoietic stem cell transplant (HSCT) recipients. Linearity, precision, and accuracy were validated over a large range of concentrations for each compound. The method could measure analytes with high sensitivity, accuracy (93-116%), and reproducibility (CV7.5%). Its clinical application was validated in PBMC lysates obtained from healthy individuals (n = 43) and HSCT recipients (n = 19). This reliable and validated LC-MS/MS method could be a useful tool for pharmacodynamic monitoring of patients treated with MMF.

Mycophenolic acid (MPA) inhibits the enzyme inosine 5'-monophosphate dehydrogenase (IMPDH). Thus, the measurement of IMPDH activity could serve as a specific pharmacodynamic (PD) tool for monitoring MPA therapy. At present, however, monitoring of pharmacokinetic parameters is preferred over that of PD parameters because, in general. PD assays are labor-intensive and poorly reproducible. Currently, cell count or protein concentration is widely accepted as methods to normalize enzyme activity. In the present study, we have attempted to further improve a method for the determination of IMPDH activity to increase the robustness and reproducibility of the IMPDH activity assay itself, without making the assay more labor-intensive. Therefore, several aspects of the IMPDH method were investigated regarding their influence on the reproducibility and also modified to increase the feasibility and consistency of the assay. The isolation of peripheral blood mononuclear cells (PBMCs) of whole blood samples was found to be the most variable step. Normalization on cell count is labor-intensive and at the same time has a poor reproducibility. Determination of the protein content in cell extracts is impaired by contamination with extracellular proteins and non-PBMCs. Alternatively, the intracellular substance adenosine monophosphate (AMP) was investigated to normalize the newly generated xanthosine monophosphate. Among various subject groups, no significant differences in mean AMP concentration were found. To simplify the procedure, PBMCs were diluted to a fixed volume after isolation from sample of whole blood, and the IMPDH activity was normalized to the AMP concentration quantified in the same high-performance liquid chromatography run as xanthosine monophosphate was quantified. The within-run and total imprecision (coefficient of variation) ranged from 4.2% to 10.6% and from 6.6% to 11.9%, respectively. In conclusion, the modified method described here for the measurement of IMPDH activity can be used reliably in multicenter trials and in longitudinal studies to evaluate the additional value of any PD monitoring among a diversity of patients treated with MPA.

The first acidity constant of fully protonated xanthosine 5'-monophosphate, that is, of H3(XMP)+, was estimated by means of a micro acidity constant scheme and the following three deprotonations of the H2(XMP)+/- (pKa=0.97), H(XMP)- (5.30), and XMP2- (6.45) species were determined by potentiometric pH titrations; further deprotonation of (XMP-H)3- is possible only with pKa>12. The most important results are that the xanthine residue is deprotonated before the P(O)2(OH)- group loses its final proton; that is, twofold negatively charged XMP carries one negative charge in the pyrimidine ring and one at the phosphate group. Micro acidity constant evaluations reveal that this latter mentioned species occurs with a formation degree of 88 %, whereas its tautomer with a neutral xanthine moiety and a PO3(2-) group is formed only to 12 %; this distinguishes XMP from its related nucleoside 5'-monophosphates, like guanosine 5'-monophosphate. At the physiological pH of about 7.5 mainly (XMP-H)3- exists. The question, which of the purine sites, (N1)H or (N3)H, is deprotonated in this species cannot be answered unequivocally, though it appears that the (N3)H site is more acidic. By application of several methylated xanthine species intrinsic micro acidity constants are calculated and it is shown that, for example, for 7-methylxanthine the N1-deprotonated tautomer occurs with a formation degree of about 5 %; a small but significant amount that, as is discussed, may possibly be enhanced by metal ion coordination to N7, which is known to occur preferably to this site.

Inosine-5′-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed “Rods and Rings” (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.

Mycophenolate mofetil (MMF) is an ester prodrug with higher bioavailability than the active agent, mycophenolic acid (MPA).1 MPA inhibits inosine monophosphate (IMP) dehydrogenase, an enzyme that facilitates the conversion of IMP to xanthosine monophosphate, a precursor of guanine nucleotides. This is an important step in the de novo pathway of purine nucleotide synthesis on which lymphocytes primarily depend, unlike neutrophils, for example.2 In addition, different isoforms of IMP dehydrogenase exist, and MPA is not only almost five times more potent in inhibiting the type II isoform, that is associated with stimulated rather than resting lymphocytes,3 but is also, to some extent, cell type specific.4 Thus, on theoretical grounds, MMF represents a significant improvement on the antimetabolite azathioprine the use of which is well-established in organ transplantation. MMF is effective in prolonging the survival of mouse and rat cardiac allografts,5,,6 showing an additive effect with cyclosporin, brequinar or tacrolimus.6–,9 It can also reverse rejection of rat cardiac allografts6 and prolong canine hepatic allograft survival10 as well as mouse11–,13 and rat14 pancreatic allograft survival. Finally, in a canine model, it has been shown to prolong renal allograft survival15 and reverse acute rejection.16 Rat allograft studies have reported a role in reducing ischaemia-reperfusion injury.17 An additional experimental observation of potential interest in organ transplantation is the inhibitory effect of MPA on the proliferation of human arterial smooth muscle cells in vitro . Arterial disease plays a central role in chronic graft rejection, holding out hope for additional benefit from the use of MMF.18 In vivo MMF inhibits the graft coronary artery disease of rat cardiac allografts19 and cynomologous monkey-to-baboon cardiac xenografts20 and it attenuates functional, morphological and immunohistochemical changes associated with … Dr A.N. Warrens, Renal Unit, Imperial College School of Medicine, Hammersmith Hospital, L Block, Du Cane Road, London W12 0HS. e-mail: a.warrens{at}ic.ac.uk

Inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) are effective immunosuppressive drugs that may also have additional potential applications as antitumour and antimicrobial agents. The clinical value of the most potent and specific inhibitor of IMPDH, mycophenolic acid, is limited by its rapid metabolism in vivo to an inactive glucuronide derivative. There is, therefore, a considerable incentive to develop structurally novel, preferably non-nucleoside, inhibitors with greater metabolic stability than mycophenolic acid. Here, we describe a high throughput screen for inhibitors of IMPDH, which facilitated the discovery of a single novel non-nucleoside inhibitor from a collection of approximately 80,000 compounds. The inhibitor is a pyridazine, which, like mycophenolic acid, exerts uncompetitive inhibition of IMPDH. Analysis of the enzyme kinetics suggests that the inhibitory action of the pyridazine is similar to that of mycophenolic acid, which involves trapping of a covalent intermediate formed during the conversion of IMP to xanthosine monophosphate. Chemical modification of the lead compound resulted in pyridazine derivatives with enhanced potency against IMPDH and guanine nucleotide synthesis in cultured cells in vitro and also against guanine nucleotide synthesis in the mouse spleen in vivo. One of the compounds was available in sufficient quantity to demonstrate highly effective immunosuppressive activity in a model of delayed type hypersensitivity in mice. To our knowledge, the novel pyridazines described in this report represent the first non-nucleoside uncompetitive inhibitors of IMPDH with immunosuppressive activity since the discovery of the inhibitory activity of mycophenolic acid and its derivatives thirty years ago.

The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k(cat)/K(m) values determined at 323 and 353 K fall between 1.31 × 10(4) and 7.80 × 10(4) M(-1) s(-1). ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg(2+) as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 A resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase).

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.

Enzymatic efficiency and structural discrimination of substrates from nonsubstrate analogues are attributed to the precise assembly of binding pockets. Many enzymes have the additional remarkable ability to recognize several substrates. These apparently paradoxical attributes are ascribed to the structural plasticity of proteins. A partially defined active site acquires complementarity upon encountering the substrate and completing the assembly. Human hypoxanthine guanine phosphoribosyltransferase (hHGPRT) catalyzes the phosphoribosylation of guanine and hypoxanthine, while the Plasmodium falciparum HGPRT (PfHGPRT) acts on xanthine as well. Reasons for the observed differences in substrate specificities of the two proteins are not clear. We used ultraviolet resonance Raman spectroscopy to study the complexes of HGPRT with products (IMP, GMP, and XMP), in both organisms, in resonance with the purine nucleobase electronic absorption. This led to selective enhancement of vibrations of the purine ring over those of the sugar-phosphate backbone and protein. Spectra of bound nucleotides show that HGPRT distorts the structure of the nucleotides. The distorted structure resembles that of the deprotonated nucleotide. We find that the two proteins assemble similar active sites for their common substrates. While hHGPRT does not bind XMP, PfHGPRT perturbs the pK(a) of bound XMP. The results were compared with the mutant form of hHGPRT that catalyzed xanthine but failed to perturb the pK(a) of XMP.

Mycophenolic acid glucuronide (MPAG) inhibition kinetics were evaluated using purified recombinant human type II inosine monophosphate dehydrogenase (IMPDH). MPAG inhibitory concentrations (IC 50 ) were found to be 532- to 1022-fold higher than those for MPA. As expected, according to tight-binding inhibitor kinetics, mycophenolic acid (MPA) IC 50 values increased as IMPDH concentrations increased, whereas IC 50 values for xanthosine monophosphate (competitive IMPDH inhibitor used as a control), an inhibitor known not to be tight binding, remained independent of enzyme concentration. Although MPAG exhibited only weak inhibition of IMPDH activity, in comparison with MPA, IC 50 values increased with increasing enzyme concentration. The presence of trace quantities of MPA (0.2% on a molar basis) in the MPAG preparation, detected by high-performance liquid chromatography analysis, could account for this observation. These data support the proposal that MPAG is a pharmacologically inactive metabolite of MPA.

Mycophenolate Mofetil (MMF) is an ester prodrug of the immunosuppressant myco-phenolic acid (MPA), which selectively inhibits the inosine monophosphate dehydrogenase (IMPDH). IMPDH converts inosine monophosphate (IMP) into xanthosine monophosphate (XMP) which will further be metabolized to ATP and GTP.

Mycophenolate Mofetil (MMF) is an ester prodrug of the immunosuppressant myco-phenolic acid (MPA), which selectively inhibits the inosine monophosphate dehydrogenase (IMPDH). IMPDH converts inosine monophosphate (IMP) into xanthosine monophosphate (XMP) which will further be metabolized to ATP and GTP.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an attractive drug target for the control of parasitic infections. The enzyme catalyzes the NAD-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the committed step in guanosine monophosphate (GMP) biosynthesis. We have determined the crystal structures of IMPDH from the protozoan parasite Tritrichomonas foetus in the apo form at 2.3 A resolution and the enzyme-XMP complex at 2.6 A resolution. The enzyme forms a cyclic (C4) homotetramer. The core domain of each monomer forms an eight-stranded parallel beta/alpha barrel with the enzyme active site at the C-termini of the barrel beta strands which lies near the center of the fourfold axis of the tetramer. While the electron-density for XMP in the complex structure is well-defined, the NAD cofactor and a nearby loop containing the catalytic cysteine (Cys-319) are disordered. This disorder at the active site suggests that a high degree of flexibility may be inherent to the catalytic function of IMPDH, making this area a difficult target for structure-based inhibitor design. Unlike IMPDHs from other species, the T. foetus enzyme coordinates the substrate phosphate with a single arginine guanidinium in the active site. Furthermore, a deep groove extends 8 A from the substrate phosphate away from the sugar. This structural uniqueness forms the basis of our efforts to design compounds that specifically inhibit the parasite enzyme.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an attractive drug target for the control of parasitic infections. The enzyme catalyzes the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the committed step in de novo guanosine monophosphate (GMP) biosynthesis. We have determined the crystal structures of IMPDH from the protozoan parasite Tritrichomonas foetus in the apo form at 2.3 A resolution and the enzyme-XMP complex at 2.6 A resolution. Each monomer of this tetrameric enzyme is comprised of two domains, the largest of which includes an eight-stranded parallel beta/alpha-barrel that contains the enzyme active site at the C termini of the barrel beta-strands. A second domain, comprised of residues 102-220, is disordered in the crystal. IMPDH is expected to be active as a tetramer, since the active site cavity is formed by strands from adjacent subunits. An intrasubunit disulfide bond, seen in the crystal structure, may stabilize the protein in a less active form, as high concentrations of reducing agent have been shown to increase enzyme activity. Disorder at the active site suggests that a high degree of flexibility may be inherent in the catalytic function of IMPDH. Unlike IMPDH from other species, the T. foetus enzyme has a single arginine that is largely responsible for coordinating the substrate phosphate in the active site. This structural uniqueness may facilitate structure-based identification and design of compounds that specifically inhibit the parasite enzyme.

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.

The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophosphate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (pur A), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.

GMP reductase was highly purified from promastigotes of Leishmania donovani by chromatography on a single DEAE-cellulose column. Bimodal substrate saturation curves resulted in a 1/v versus 1/[GMP] plot that curved downward above 40 microM GMP. The kinetic constants were, therefore, obtained with GMP below this concentration. The K'm for GMP was 21 microM at pH 6.9. The enzyme was very sensitive to activation by GTP. At 20 microM GMP, a maximum of 600% activation occurred at 100 microM GTP. Half-maximal activation occurred at 8 microM GTP. GTP at 100 microM did not affect the K'm for GMP but did increase its V'max by 7-fold. Xanthosine monophosphate (XMP) and IMP analogs served equally well as competitive inhibitors versus GMP. The inhibition by the analogs and the activation by GTP were mutually antagonistic processes. The inhibition by the IMP analogs, allopurinol nucleotide and thiopurinol nucleotide is of chemotherapeutic interest because these compounds were shown previously to be produced in Leishmania from the anti-leishmanial agents allopurinol and thiopurinol. These nucleotides were 100- and 20-fold, respectively, more potent inhibitors of GMP reductase from L. donovani than of the corresponding enzyme from human erythrocytes.

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.

1. 5′-Nucleotidase activity was obtained in a soluble form after treatment of a particulate fraction from Ehrlich ascites-tumour cells with deoxycholate. The relative rates of hydrolysis of 6-thioinosine 5′-phosphate, UMP, AMP, CMP, GMP, IMP, xanthosine monophosphate, thymidine monophosphate and 2′,3′-AMP were 180, 129, 100, 93, 83, 79, 46, 41 and 3 respectively. 2. Values found for the Michaelis constant were: AMP, 67±12μm; IMP, 111±8μm; GMP, 93μm. 3. ATP and thymidine triphosphate were competitive inhibitors of AMP hydrolysis (inhibitor constants 0·4 and 4·8μm respectively); UTP, GTP and CTP were mixed competitive and non-competitive inhibitors. Thymidine triphosphate was a competitive inhibitor of IMP hydrolysis (inhibitor constant 14·4μm) and ATP, UTP and GTP showed mixed competitive and non-competitive inhibition. 4. ATP, thymidine triphosphate, UTP, GTP and CTP did not completely inhibit hydrolysis of AMP, IMP and UMP; the concentrations of ATP required to inhibit AMP and IMP hydrolysis by 50% were 12 and 230μm respectively. 5. Non-hyperbolic curves relating activity to UMP concentration were obtained in the presence and absence of triphosphates. 6. After fractionation on Sephadex G-200 columns a single peak of 5′-nucleotidase activity (particle weight 120000–125000) was obtained with AMP, IMP and GMP as substrates. UMP hydrolysis was catalysed by enzyme in this peak and in two slower peaks corresponding to apparent particle weights of 32000 and 16000; a single component (particle weight 120000), reacting with UMP and insensitive to UTP inhibition, was obtained when the column was eluted with buffer containing 1mm-UMP. 7. The possible significance of the results in the regulation of tumour-cell 5′-nucleotidase is discussed.

We have investigated the changes in the guanosine 5′-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5′-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5′-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5′-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.

Inosine monophosphate dehydrogenase (IMPD) is an important enzyme in de-novo purine synthesis. The level of IMPD activity has been suggested to determine whether acute leukaemia cells proliferate (if the activity is high) or differentiate (if IMPD activity is low). IMPD activity measured by the conversion of inosine monophosphate to xanthine monophosphate ranged from 12.5 to 87.0 (mean 49.4) pmol/h/10 6 cells in normal bone marrow. The levels were significantly raised in AML (range 14–374, mean 184 pmol/h/10 6 cells) and ALL (range 65–228, mean 172 pmol/h/10 6 cells). Normal tonsillar (B) lymphocytes showed higher levels (range 78–159, mean 110 pmol/h/10 6 cells) than resting peripheral blood T lymphocytes (range 8.8–51.2, mean 28.1 pmol/h/10 6 cells). In CLL, the results (range 19–173, mean 64.3 pmol/h/10 6 cells) were comparable to those of normal tonsillar B lymphocytes. IMPD levels could be related to cell cycle in PHA-stimulated lymphocytes, since IMPD activity increased in parallel with increase in DNA synthesis measured by labelled thymidine incorporation. On the other hand, IMPD activity did not correlate with the proportion of proliferating cells measured on a FACS sorter in either AML or ALL or in normal tonsillar B cells. We conclude that IMPD levels are higher in B than T lymphocytes and in acute leukaemia blasts compared to more differentiated mixed bone marrow cells. The results do not suggest, however, that IMPD assay will be of value in differentiation of the various subtypes of acute leukaemia or of malignant haemopoietic cells from the equivalent normal cell at the same level of differentiation.

Publisher Summary This chapter discusses guanine monphosphate (GMP) synthetase from ehrlich ascites cells. Four assays were developed to meet the special requirements of these studies. This chapter describes all these assays. Assays I–III couple the formation of adenosine monophosphate (AMP) and/or GMP to the oxidation of NADH as mediated through AMP kinase and/or GMP kinase, pyruvate kinase, and lactate dehydrogenase. The oxidation is monitored spectrophotometrically at 340 or 360 nm. Assay I measures the formation of AMP and, therefore, has the advantage of being nonspecific with respect to the substrate undergoing amination. Assay II measures GMP formation and is, therefore, useful for selectively monitoring the amination of Xanthosine monophosphate (XMP). Assay III combines assays I and II to produce a doubling of the sensitivity. Assay IV is radiochemical and can be performed in volumes as small as 50μl. GMP synthetase obtained from either of the above described column steps can be fast-frozen and stored at -70° for at least one year with no loss of activity. Thawing and refreezing do not diminish the activity.

Publisher Summary A variety of human and animal diseases are caused by parasitic protozoa including malaria, chagas disease (South American trypanosomiasis), african trypanosomiasis, giardiasis, leishmaniasis, coccidiosis, and toxoplasmosis. The nudeotide metabolism of these organisms often differs strikingly from the metabolism of the host mammalian cells. This chapter covers some of the major chemotherapeutic studies carried out on parasitic protozoa recently in the area of purine or pyrimidine nucleotide metabolism. The designation R after a compound number stands for β -D-riboside, RP for the 5' monophosphate and RP 3 , for the 5' triphosphate. This chapter discusses pyraaolopyrimidine analogs, inosine analogs, Formyun B, thiopuinol analogs, inhibition of guanine nucleotide formation, thymidylate synthetase-dihydrofolate reductase, deoxynucleoside phosphotransferase, adenosine deaminase, GMP reductase, and guanine aminohydrolase. Derivatives of pyraaolopyrimidine, including the parent compound albpcrinol, have been intensively studied because they are metabolized in an unusual lay by pathogenic parasitic protozoa of the genera Leishmania and Trypanosoma, which results in antiparasitic activity. The parasite has high levels of hypxanthine guanine phophoribosyl transferase, HCPRTase, which phosphoribosylates the purine analog, and also lacks xanthine oxidase to catalyze oxidation to oxypuinol, which occurs readily in mammalian cells. The succino-AMP synthetase and sucuno-AMP lyase, which normally carry out the conversion of IMP to AMP, have been shown to convert 1-RP to 2-RP if the enzyme from Leishmania donovani or Tranosoma cruzi is used, whereas no conversion occurs with the mammalian enzyme. Guanine nucleotide production in parasitic protoma has been examined as a target in several parasitic protoma. IMP dehydrogenase is responsible for the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), from which CMP is made. Inhibition of this enzyme using mycophenolic acid decreased guanine nucleotide synthesis in Leishmania tropica promastigotes.

Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion. Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown. Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside. Adenosine, inosine, xanthosine, and guanosine were converted to their base. No ATP-dependent phosphorylation of nucleosides to mononucleotides was found. However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected. Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found. Interconversion of GMP to AMP was detected. Enzyme activities for the interconversion of AMP to GMP were not detected. Therefore, A. laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates. De novo synthesis of purines was not detected. This study demonstrates that A. laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp. mycoides. This is the first report of a PPi-dependent nucleoside kinase activity in any organism.

Corynebacterium species lacks the ability to convert either xanthine or guanine to adenine. This defect and the use of the purine nucleoside antibiotic decoyinine, which blocks the conversion of xanthosine monophosphate → guanosine monophosphate, permit an experimental design in which the interconversion of purines is largely prevented. Cultures of this organism were grown in the presence of decoyinine and various purine supplements. Data obtained by comparing the radioactivity incorporated from guanine- 2 - 14 C or xanthine- 2 - 14 C into bacterial guanine, xanthine, and riboflavine indicate that guanine or a close derivative of guanine is the purine precursor of riboflavine.

— An investigation of the methylene blue sensitized oxidation of isolated nucleotides has been carried out, based on the behaviour of the excited dye molecule in long-term irradiation and flash photolysis experiments. It was necessary first to establish the effect of the phosphate buffer upon the triplet excited state of the dye and the consequent effect upon its photo-fading. The buffer has a salt effect which accelerates the protonation of the dye triplet, and also the photoreduction of the dye which takes place through the resulting protonated triplet. Xanthosine monophosphate (XMP), which is the nucleotide most sensitive to the photodynamic action of methylene blue, is also effective in reducing the triplet of the dye, while other nucleotides which are resistant to photooxidation do not act as reducing agents. The quantum yield of the sensitized oxidation of XMP was found to correspond to that of the anaerobic photo-reduction of methylene blue in its presence, which leads to the conclusion that the primary step of the photooxidation is electron or hydrogen abstraction from the nucleotide by the triplet excited methylene blue molecule.