Guanosine Monophosphate Synthetase Research Articles

Advances in human glutamine-hydrolyzing synthetases and their therapeutic potential

Bifunctional enzymes, characterized by their dual active sites, enable efficient chemical conversion and substrate channeling using elegant coupling mechanisms to coordinate the two active sites. In humans, several bifunctional enzymes synthesize de novo carbon-nitrogen bonds by hydrolyzing glutamine and ATP in distinct active sites. Notable examples include guanosine monophosphate synthetase, cytidine triphosphate synthetase, phosphoribosylformyl-glycinamidine synthase, asparagine synthetase, and nicotinamide adenine dinucleotide synthetase. A more complex example of multifunctional glutamine-hydrolyzing synthetases in humans is carbamoyl phosphate synthetase. These enzymes are crucial for the biosynthesis of amino acids, nucleic acids, and co-factors, thereby playing pivotal roles in human health. This review delineates recent progress in understanding the structural characteristics, regulatory mechanisms, and disease relevance of glutamine-hydrolyzing synthetases in humans. Insights into their catalysis and activity regulation offer potential pathways for developing novel therapeutics.

GMP Synthetase: Allostery, Structure, and Function.

Glutamine amidotransferases (GATs) catalyze the hydrolysis of glutamine and transfer the generated ammonia to diverse metabolites. The two catalytic activities, glutaminolysis and the subsequent amination of the acceptor substrate, happen in two distinct catalytic pockets connected by a channel that facilitates the movement of ammonia. The de novo pathway for the synthesis of guanosine monophosphate (GMP) from xanthosine monophosphate (XMP) is enabled by the GAT GMP synthetase (GMPS). In most available crystal structures of GATs, the ammonia channel is evident in their native state or upon ligand binding, providing molecular details of the conduit. In addition, conformational changes that enable the coordination of the two catalytic chemistries are also informed by the available structures. In contrast, despite the first structure of a GMPS being published in 1996, the understanding of catalysis in the acceptor domain and inter-domain crosstalk became possible only after the structure of a glutamine-bound mutant of Plasmodium falciparum GMPS was determined. In this review, we present the current status of our understanding of the molecular basis of catalysis in GMPS, becoming the first comprehensive assessment of the biochemical function of this intriguing enzyme.

Biology of Two-Spotted Spider Mite (Tetranychus urticae): Ultrastructure, Photosynthesis, Guanine Transcriptomics, Carotenoids and Chlorophylls Metabolism, and Decoyinine as a Potential Acaricide.

Two-Spotted Spider Mites (TSSMs, Tetranychus urticae Koch 1836 (Acari: Tetranychidae)) is one of the most important pests in many crop plants, and their feeding activity is based on sucking leaf cell contents. The purpose of this study was to evaluate the interaction between TSSMs and their host Lima bean (Phaseolus lunatus) by analyzing the metabolomics of leaf pigments and the transcriptomics of TSSM guanine production. We also used epifluorescence, confocal laser scanning, and transmission electron microscopies to study the morphology and structure of TSSMs and their excreta. Finally, we evaluated the potential photosynthetic ability of TSSMs and the activity and content of Ribulose-1,5-bisphosphate Carboxylase/Oxigenase (RubisCO). We found that TSSMs express several genes involved in guanine production, including Guanosine Monophosphate Synthetase (GMPS) and decoyinine (DCY), a potential inhibitor of GMPS, was found to reduce TSSMs proliferation in infested Lima bean leaves. Despite the presence of intact chloroplasts and chlorophyll in TSSMs, we demonstrate that TSSMs do not retain any photosynthetic activity. Our results show for the first time the transcriptomics of guanine production in TSSMs and provide new insight into the catabolic activity of TSSMs on leaf chlorophyll and carotenoids. Finally, we preliminary demonstrate that DCY has an acaricidal potential against TSSMs.

Comprehensive characterization of Guanosine monophosphate synthetase in Nicotiana tabacum.

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.

Inhibition of guanosine monophosphate synthetase (GMPS) blocks glutamine metabolism and prostate cancer growth.

Glutamine is a critical nutrient in cancer; however, its contribution to purine metabolism in prostate cancer has not previously been determined. Guanosine monophosphate synthetase (GMPS) acts in the de novo purine biosynthesis pathway, utilizing a glutamine amide to synthesize the guanine nucleotide. This study demonstrates that GMPS mRNA expression correlates with Gleason score in prostate cancer samples, while high GMPS expression was associated with decreased rates of overall and disease/progression-free survival. Pharmacological inhibition or knockdown of GMPS significantly decreased cell growth in both LNCaP and PC-3 prostate cancer cells. We utilized [15 N-(amide)]glutamine and [U-13 C5 ]glutamine metabolomics to dissect the pathways involved and despite similar growth inhibition by GMPS knockdown, we show unique metabolic effects across each cell line. Using a PC-3 xenograft mouse model, tumor growth was also significantly decreased after GMPS knockdown, highlighting the importance of glutamine metabolism and providing support for GMPS as a therapeutic target in prostate cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Conserved bacterial de novo guanine biosynthesis pathway enables microbial survival and colonization in the environmental niche of the urinary tract.

In bacteria, guaA encodes guanosine monophosphate synthetase that confers an ability to biosynthesize guanine nucleotides de novo. This enables bacterial colonization in different environments and, while guaA is widely distributed among Bacteroidetes and Firmicutes, its contribution to the inhabitation of the human microbiome by commensal bacteria is unclear. We studied Streptococcus as a commensal urogenital tract bacterium and opportunistic pathogen, and explored the role of guaA in bacterial survival and colonization of urine. Analysis of guaA-deficient Streptococcus revealed guanine utilization is essential for bacterial colonization of this niche. The genomic location of guaA in other commensals of the human urogenital tract revealed substantial cross-phyla diversity and organizational structures of guaA that are divergent across phyla. Essentiality of guaA for Streptococcus colonization in the urinary tract establishes that purine biosynthesis is a critical element of the ability of this bacterium to survive and colonize in the host as part of the resident human microbiome.

Association mapping for traits related to nitrogen use efficiency in tropical maize lines under field conditions

Nitrogen deficiency is one of the most critical abiotic stresses for maize (Zea mays) cultivation worldwide. For a productive and sustainable scenario, developing genotypes more efficient in nitrogen use is essential. This study aimed to identify single nucleotide polymorphism (SNP) markers associated with nitrogen use efficiency (NUE) traits under field conditions and candidate genes related to these markers by genome-wide association study (GWAS). Sixty-four tropical maize inbred lines were evaluated in ideal and low nitrogen conditions for total root length (TRL) and low nitrogen tolerance index (LNTI). GWAS was performed using a fixed and random model circulating probability unification method, with marker-based principal components to correct for population stratification. Genotypic values were predicted using mixed model equations. Seven significant markers were identified. Among the primary biological processes, candidate genes are related to transcription control and regulation, detected to all evaluated traits, and the synthesis of Guanosine Monophosphate Synthetase, enzyme directly involved in the provision and recycling of nitrogen. GWAS analysis revealed genomic regions in tropical maize associated with NUE under field conditions. The main biological process identified as related to these markers/regions evidence cellular processes and functions associated with different process of nitrogen synthesis and recycling.

P38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation. DisclosuresNo relevant conflicts of interest to declare.

Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for Mycobacterium tuberculosis growth

Guanosine monophosphate synthetase (GMPS), encoded by guaA gene, is a key enzyme for guanine nucleotide biosynthesis in Mycobacterium tuberculosis. The guaA gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct guaA deletion in M. tuberculosis has been described so far. Here, we demonstrated that the guaA gene is essential for M. tuberculosis H37Rv growth. The lethal phenotype of guaA gene disruption was avoided by insertion of a copy of the ortholog gene from Mycobacterium smegmatis, indicating that this GMPS protein is functional in M. tuberculosis. Protein validation of the guaA essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from M. tuberculosis and M. smegmatis GMPS proteins. These results validate GMPS as a molecular target for drug design against M. tuberculosis, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.

Homeostatic generation of reactive oxygen species protects the zebrafish liver from steatosis

Nonalcoholic fatty liver disease is the most common liver disease in both adults and children. The earliest stage of this disease is hepatic steatosis, in which triglycerides are deposited as cytoplasmic lipid droplets in hepatocytes. Through a forward genetic approach in zebrafish, we found that guanosine monophosphate (GMP) synthetase mutant larvae develop hepatic steatosis. We further demonstrate that activity of the small GTPase Rac1 and Rac1-mediated production of reactive oxygen species (ROS) are down-regulated in GMP synthetase mutant larvae. Inhibition of Rac1 activity or ROS production in wild-type larvae by small molecule inhibitors was sufficient to induce hepatic steatosis. More conclusively, treating larvae with hydrogen peroxide, a diffusible ROS that has been implicated as a signaling molecule, alleviated hepatic steatosis in both GMP synthetase mutant and Rac1 inhibitor-treated larvae, indicating that homeostatic production of ROS is required to prevent hepatic steatosis. We further found that ROS positively regulate the expression of the triglyceride hydrolase gene, which is responsible for the mobilization of stored triglycerides in hepatocytes. Consistently, inhibition of triglyceride hydrolase activity in wild-type larvae by a small molecule inhibitor was sufficient to induce hepatic steatosis. De novo GMP synthesis influences the activation of the small GTPase Rac1, which controls hepatic lipid dynamics through ROS-mediated regulation of triglyceride hydrolase expression in hepatocytes.

Solution Nuclear Magnetic Resonance Structure of the GATase Subunit and Structural Basis of the Interaction between GATase and ATPPase Subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500mg). Over the 44h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2wk before starting PC1 treatment. Bacterial counts reached then about 103 to 104 cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Inhibition of Guanosine Monophosphate Synthetase by the Substrate EnantiomerL-XMP

Studies with mirror-image l-enantiomer nucleosides and nucleotides have revealed relaxed enantioselectivities of several cellular kinases and viral polymerases.[1,2] This feature of enzyme-ligand molecular recognition has been exploited in the design of efficacious antiviral l-nucleoside drugs, which have lowered host cell toxicity.[3-6] For example, lamivudine (2′,3′-dideoxy-3′-thiacytidine, 3TC), an l-nucleoside drug, exploits the relaxed enantioselectivity of HIV reverse transcriptase to inhibit viral replication.[7] Conversely, the enantioselectivities of the majority of nucleotide biosynthesis enzymes have not been characterized. The depletion of cellular nucleotide pools has been shown to result in antiproliferative, antibacterial, and immunosuppressive effects.[8-11]

1H, 13C, 15N assignment and secondary structure determination of glutamine amido transferase subunit of gaunosine monophosphate synthetase from Methanocaldococcus jannaschii

Sequence specific resonance assignments have been obtained for (1)H, (13)C and (15)N nuclei of the 21 kDa (188 residues long) glutamine amido transferase subunit of guanosine monophosphate synthetase from Methanocaldococcus jannaschii. From an analysis of (1)H and (13)C(α), (13)C(β) secondary chemical shifts, (3) JH(N)H(α) scalar coupling constants and sequential, short and medium range (1)H-(1)H NOEs, it was deduced that the glutamine amido transferase subunit has eleven strands and five helices as the major secondary structural elements in its tertiary structure.

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.

The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells

Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP- and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122- and 93-fold higher in 6-TG- and 6-MP-resistant cells, respectively. The varying responses to 6-MP- and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.

Allopurinol Salvage Therapy in Pediatric Overlap Autoimmune Hepatitis‐Primary Sclerosing Cholangitis With 6‐MMP Toxicity

Allopurinol Salvage Therapy in Pediatric Overlap Autoimmune Hepatitis‐Primary Sclerosing Cholangitis With 6‐MMP Toxicity

Azathioprine and allopurinol: A two‐edged interaction

The thiopurines, azathioprine and 6-mercaptopurine (6-MP), are immunosuppressive drugs used in a number of clinical settings, such as following transplantation and for the management of inflammatory conditions like inflammatory bowel disease (IBD). Allopurinol, a xanthine oxidase inhibitor used in the treatment of gout, is not infrequently coincidentally co-prescribed with the thiopurines. A recent safety report from the New South Wales Department of Health (NSW, Australia) highlights the potential risk with the co-administration of these two drugs. This commentary reviews the interaction of these drugs and highlights important precautions that should be taken by clinicians when using them together.

Genetic Variations in the HGPRT, ITPA, IMPDH1, IMPDH2, and GMPS Genes in Japanese Individuals

Thiopurines (such as azathioprine and 6-mercaptopurine) are widely used for the treatment of patients suffering from malignancies, rheumatic disease, inflammatory bowel disease and solid organ transplant rejection. These drugs are activated and eliminated by a number of enzymes in the human body. This analyzes all the exons and exon-intron junctions of 5 enzyme genes (hypoxanthine-guanine phosphoribosyltransferase, HGPRT; inosine triphosphate pyrophosphatase, ITPA; inosine monophosphate dehydrogenases 1 and 2, IMPDH1 and IMPDH2 and guanosine monophosphate synthetase, GMPS) involved in the metabolism of thiopurine drugs. Twelve novel single nucleotide polymorphisms (SNPs) (HGPRT: IVS6-12C>A (frequency:0.003); ITPA: 569T>C (Phe189Phe, 0.003); IMPDH1: IVS8-15C>A (0.003), IVS9+227A>G (0.003), IVS17+115C>T (0.003), and 930C>T (Thr310Thr, 0.005); IMPDH2: IVS1+50G>T (0.003), IVS2+15G>A (0.010), IVS3-20G>A (0.003), 609C>T (Arg203Arg, 0.003), and 1534C>T (Arg512Trp, 0.003); and GMPS: 1563T>C (Gly521Gly, 0.003)) and 7 known SNPs (ITPA: 94C>A (Pro32Thr, 0.005), 138G>A (Gln46Gln, 0.586), and 563G>A (Glu187Glu, 0.433); IMPDH1: 987G>C (Leu329Leu, 0.113) and 1575A>G (Ala525Ala, 0.620) and GMPS: IVS5-7T>C (0.153), 993A>G (Thr331Thr, 0.153)) were identified in 200 Japanese subjects. These data should provide useful information for thiopurine therapy in the Japanese and as well as other Asian populations.

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28 kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa ( Plasmodium, Babesia, Toxoplasma) or zoomastigina ( Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.