GMP Synthetase Research Articles

Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, here we report that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. Our data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.

Tertiary and Quaternary Structure Organization in GMP Synthetases: Implications for Catalysis

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH3 generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis.

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase.

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

Mechanism of allostery and catalysis in P. falciparum GMP Synthetase

Mechanism of allostery and catalysis in <i>P. falciparum</i> GMP Synthetase

Proteomic Analysis Reveals GMP Synthetase as p53 Repression Target in Liver Cancer

Disruption of the tumor-suppressive p53 network is a key event in human malignancies, including primary liver cancer. In response to different types of stress, p53 mediates several antiproliferative cellular outcomes, such as cell cycle arrest, apoptosis, and senescence, by activation or repression of its target genes. Metabolic alterations initiating or being part of the p53 response have become an actively studied research area in the p53 field, with several aspects that still remain to be elucidated. Herein, we identified GMP synthetase (GMPS), a key enzyme of de novo purine biosynthesis, as an important p53 repression target using a large-scale proteomics approach. This p53-mediated repression of GMPS could be validated by immunoblotting in Sk-Hep1, HepG2, and HuH6 cells. Moreover, we found GMPS transcriptionally repressed in a p21-dependent manner and its repression maintained in the context of p53-mediated cellular senescence. More important, direct knockdown of GMPS by RNA interference resulted in reduced cell viability and was sufficient to trigger cellular senescence. Finally, by comparing murine hepatocellular carcinomas, which developed in p53 wild-type (+/+) versus p53 null (-/-) mice, we observed higher GMPS expression in the latter, supporting the invivo relevance of our findings. We conclude that repression of GMPS by p53 through p21 is a functionally relevant part of the p53-mediated senescence program limiting tumor cell growth in liver cancer.

Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase.

Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii. By comparing the biophysical properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability.

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371–375 holding catalytic residues and in loop 376–401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme.

Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling

Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers.

Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase

GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.

The role of UBL domains in ubiquitin-specific proteases

Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.

1H, 13C, 15N assignment and secondary structure determination of GATase subunit of GMP Synthetase from Methanococcus jannaschii

1H, 13C, 15N assignment and secondary structure determination of GATase subunit of GMP Synthetase from Methanococcus jannaschii

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PPi, and glutamate. XMP, NH4+, and Mg2+ displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH3 and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PPi is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.

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.

Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes

Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors.

EBNA1-Mediated Recruitment of a Histone H2B Deubiquitylating Complex to the Epstein-Barr Virus Latent Origin of DNA Replication

The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.

Antagonistic gene control through histone H2A and H2B ubiquitylation

Maintenance of gene transcription status plays a critical role in development and disease. Here, we will discuss the antagonistic functions of histone H2A and H2B ubiquitylation. Whereas histone H2A ubiquitylation is associated with gene silencing, histone H2B ubiquitylation correlates with transcription activation. We recently identified dRAF, a novel Polycomb group silencing complex that harbors dKDM2 as a key dRAF subunit that plays a pivotal role in a trans‐histone pathway involving the removal of an active histone H3 methyl mark and formation of the repressive H2Aub mark during PcG silencing. New results on the functioning of dRAF and related complexes will be presented. In addition, we will discuss developmental gene regulation by the GMP synthetase (GMPS) and Ubiquitin Specific Protease 7 (USP7), which directs histone H2Bub de‐ubiquitylation. Both the metabolic enzyme GMPS and the DUB USP7 play remarkably specific roles in the transcriptional regulation of neural development of the optical system. We will discuss the impact of histone ubiquitylation on gene‐specific transcription control.

Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman.

A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface-enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface-enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or as HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface-enhanced resonance Raman scatter to quantification of protein distributions.