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The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His 6 -tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His 6 -tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

Adenylosuccinate synthetase (PurA) is an enzyme responsible for the nitrogen addition to inosine monophosphate (IMP) by aspartate in the purine nucleotide biosynthetic pathway. And after which the fumarate is removed by adenylosuccinate lyase (PurB), leaving an amino group. There are two other enzymes that catalyze aspartate addition reactions similar to PurA, one in the purine nucleotide biosynthetic pathway (SAICAR synthetase, PurC) and the other in the arginine biosynthetic pathway (argininosuccinate sythetase, ArgG). To investigate the origin of these nitrogen-adding enzymes, PurA from Thermus thermophilus HB8 (TtPurA) was purified and crystallized, and crystal structure complexed with IMP was determined with a resolution of 2.10 Å. TtPurA has a homodimeric structure, and at the dimer interface, Arg135 of one subunit interacts with the IMP bound to the other subunit, suggesting that IMP binding contributes to dimer stability. The different conformation of His41 side chain in TtPurA and EcPurA suggests that side chain flipping of the His41 might play an important role in orienting γ-phosphate of GTP close to oxygen at position 6 of IMP, to receive the nucleophilic attack. Moreover, through comparison of the three-dimensional structures and active sites of PurA, PurC, and ArgG, it was suggested that the active sites of PurA and PurC converged to similar structures for performing similar reactions.

Adenylosuccinate synthetase (AdSS, EC.6.3.4.4) is a key enzyme in the de novo synthesis of purine nucleotides in organisms. Its downstream product AMP plays a critical role in the process of energy metabolism, which can affect the content of ADP and ATP. However, impacts of its loss-of-function on plant metabolism and development has been relatively poorly reported. Here, we report the identification and analysis of a maize yu18 mutant obtained by mutagenesis with ethylmethane sulfonate (EMS). The yu18 is a lethal-seed mutant. Map-based cloning and allelic testing confirmed that yu18 encodes adenylosuccinate synthetase and was named ZmAdSS1. ZmAdSS1 is constitutively expressed. In the yu18 mutant, the activity of the ZmAdSS1 enzyme was decreased, which caused AMP content reduced 33.62%. The yu18 mutation significantly suppressed endoreduplication and disrupted nutrient accumulation, resulting in lower starch and protein contents that are responsible for seed filling. Further transcriptome and metabolome analysis revealed dramatic alterations in the carbohydrate metabolic pathway and amino acid metabolic pathway in yu18 kernels. Our findings demonstrate that ZmAdSS1 participates in the synthesis of AMP and affects endosperm development and nutrient accumulation in maize seeds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA., (© 2021. The Author(s).)

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. Diaminopurine (Z) is an exception because it completely replaces adenine and forms three hydrogen bonds with thymine in cyanophage S-2L genomic DNA. However, the biosynthesis, prevalence, and importance of Z genomes remain unexplored. Here, we report a multienzyme system that supports Z-genome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables Z-DNA production on a large scale for a diverse range of applications., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential., Competing Interests: Declaration of Interests The authors declare no conflicting interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To elucidate the prevalence of Japanese ADSSL1 myopathy and determine the clinicopathologic features of the disease., Methods: We searched for ADSSL1 variants in myopathic patients from January 1978 to March 2019 in our repository and assessed the clinicopathologic features of patients with variants., Results: We identified 63 patients from 59 families with biallelic variants of ADSSL1 . Among the 7 distinct variants identified, c.781G>A and c.919delA accounted for 53.2% and 40.5% of alleles, respectively, suggesting the presence of common founders, while the other 5 were novel. Most of the identified patients displayed more variable muscle symptoms, including symptoms in the proximal and/or distal leg muscles, tongue, masseter, diaphragm, and paraspinal muscles, in adolescence than previously reported patients. Dysphagia with masticatory dysfunction developed in 26 out of 63 patients; hypertrophic cardiomyopathy developed in 12 out of 48 patients; and restrictive ventilatory insufficiency developed in 26 out of 34 patients in later stages. Radiologically, fat infiltration into the periphery of vastus lateralis, gastrocnemius, and soleus muscles was observed in all patients. Pathologically, nemaline bodies in addition to increased lipid droplets and myofibrillar disorganization were commonly observed in all patients, suggesting that the disease may be classified as nemaline myopathy. This finding revealed that ADSSL1 myopathy is the most frequent among all genetically diagnosable nemaline myopathies in our center., Conclusions: ADSSL1 myopathy is characterized by more variable manifestations than previously reported. It is the most common among all genetically diagnosable nemaline myopathies in our center, although mildly increased lipid droplets are also constantly observed features., (© 2020 American Academy of Neurology.)

Adenosine, which is produced mainly by microbial fermentation, plays an important role in the therapy of cardiovascular disease and has been widely used as an antiarrhythmic agent. In this study, guanosine 5'-monophosphate (GMP) synthetase gene (guaA) was inactivated by gene-target manipulation to increase the metabolic flux from inosine 5'-monophosphate (IMP) to adenosine in B. subtilis A509. The resulted mutant M3-3 showed an increased adenosine production from 7.40 to 10.45 g/L, which was further enhanced to a maximum of 14.39 g/L by central composite design. As the synthesis of succinyladenosine monophosphate (sAMP) from IMP catalysed by adenylosuccinate synthetase (encoded by purA gene) is the rate-limiting step in adenosine synthesis, the up-regulated transcription level of purA was the potential underlying mechanism for the increased adenosine production. This work demonstrated a practical strategy for breeding B. subtilis strains for industrial nucleoside production., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a human commensal but also has devastating potential as an opportunistic pathogen. S. aureus bacteremia is often associated with an adverse outcome. To identify potential targets for novel control approaches, we have identified S. aureus components that are required for growth in human blood. An ordered transposon mutant library was screened, and 9 genes involved specifically in hemolysis or growth on human blood agar were identified by comparing the mutants to the parental strain. Three genes ( purA , purB , and pabA ) were subsequently found to be required for pathogenesis in the zebrafish embryo infection model. The pabA growth defect was specific to the red blood cell component of human blood, showing no difference from the parental strain in growth in human serum, human plasma, or sheep or horse blood. PabA is required in the tetrahydrofolate (THF) biosynthesis pathway. The pabA growth defect was found to be due to a combination of loss of THF-dependent dTMP production by the ThyA enzyme and increased demand for pyrimidines in human blood. Our work highlights pabA and the pyrimidine salvage pathway as potential targets for novel therapeutics and suggests a previously undefined role for a human blood factor in the activity of sulfonamide antibiotics., (Copyright © 2017 Connolly et al.)

To understand the characteristics of ADSSL1 myopathy, we investigated the clinical manifestation in Korean patients with ADSSL1 mutations. We developed a targeted panel of 16 distal-myopathy genes and recruited a total of 12 patients with genetically undetermined distal myopathy. We found four (33%) with ADSSL1 mutations and one (8%) with GNE mutations. ADSSL1 mutations consisted of c.910G>A, c.1048delA and c.1220T>C mutations. Patients with ADSSL1 mutations demonstrated distal muscle weakness in adolescence, followed by quadriceps muscle weakness in the early 30s. All patients had mild facial weakness and two patients complained of easy fatigue while eating and chewing. Vastus lateralis muscle biopsies revealed non-specific chronic myopathic features with a few nemaline rods. Whole body muscle MR imaging showed more fatty replacement in the distal limb and tongue muscles than in the proximal limb and axial muscles. This study showed that ADSSL1 myopathy was not rare among distal myopathy patients of Korean origin, and expanded the clinical and genetic spectrum. Therefore, we suggest that the screening test of ADSSL1 gene should be considered for the diagnosis of distal myopathy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Distal myopathy is a heterogeneous group of muscle diseases characterized by predominant distal muscle weakness. A study was done to identify the underlying cause of autosomal recessive adolescent onset distal myopathy., Methods: Four patients from 2 unrelated Korean families were evaluated. To isolate the genetic cause, exome sequencing was performed. In vitro and in vivo assays using myoblast cells and zebrafish models were performed to examine the ADSSL1 mutation causing myopathy pathogenesis., Results: Patients had an adolescent onset distal myopathy phenotype that included distal dominant weakness, facial muscle weakness, rimmed vacuoles, and mild elevation of serum creatine kinase. Exome sequencing identified completely cosegregating compound heterozygous mutations (p.D304N and p.I350fs) in ADSSL1, which encodes a muscle-specific adenylosuccinate synthase in both families. None of the controls had both mutations, and the mutation sites were located in well-conserved regions. Both the D304N and I350fs mutations in ADSSL1 led to decreased enzymatic activity. The knockdown of the Adssl1 gene significantly inhibited the proliferation of mouse myoblast cells, and the addition of human wild-type ADSSL1 reversed the reduced viability. In an adssl1 knockdown zebrafish model, muscle fibers were severely disrupted, which was evaluated by myosin expression and birefringence. In these conditions, supplementing wild-type ADSSL1 protein reversed the muscle defect., Interpretation: We suggest that mutations in ADSSL1 are the novel genetic cause of the autosomal recessive adolescent onset distal myopathy. This study broadens the genetic and clinical spectrum of distal myopathy and will be useful for exact molecular diagnostics., (© 2015 American Neurological Association.)

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Inosine and guanosine monophosphate nucleotides are convenient sources of the umami flavor, with attributed beneficial health effects that have renewed commercial interest in nucleotide fermentations. Accordingly, several bacterial strains that excrete high levels of inosine and guanosine nucleosides are currently used in the food industry for this purpose., Results: In the present study, we show that the filamentous fungus Ashbya gossypii, a natural riboflavin overproducer, excretes high amounts of inosine and guanosine nucleosides to the culture medium. Following a rational metabolic engineering approach of the de novo purine nucleotide biosynthetic pathway, we increased the excreted levels of inosine up to 27-fold., Conclusions: We generated Ashbya gossypii strains with improved production titers of inosine and guanosine. Our results point to Ashbya gossypii as the first eukaryotic microorganism representing a promising candidate, susceptible to further manipulation, for industrial nucleoside fermentation.

Thiazolidinediones (TZDs) including rosiglitazone (RSG) and pioglitazone (PIO) are synthetic agonists selective for peroxisome proliferator-activated receptor-γ (PPARγ) and have been clinically used to treat type-II diabetes as insulin sensitizers. Recent meta-analyses have shown that TZDs are associated with an increased risk for the development of heart failure. To elucidate the mechanism underlying such a cardiac adverse effect, we used a (1)H NMR-based approach to examine the metabonomic profiles in the cardiac tissues treated with RSG (15 mg/kg body weight/day) or PIO (45 mg/kg/day) for 4 weeks and found that the TZD treatments resulted in a significantly altered metabolic profile in hearts, which was associated with cardiac hypertrophy. Multivariate analysis demonstrated that TZDs led to an accumulation in adenosine monophosphate (AMP) and a depletion of inosine. Consistently, AMP kinase, a signal pathway sensitive to the change in the intracellular concentrations of AMP, was activated in the cardiac tissues from the TZDs-treated rats. Quantitative real-time reverse-transcriptase polymerase chain reaction showed a significant induction of the genes involved in the de novo synthesis of purine nucleotide but a reduction of those for the catabolism. Furthermore, the putative PPAR-responsive elements were identified in the 5'-flanking regions of the TZD-up-regulated genes such as adenylosuccinate synthase gene (Adss) and phosphoribosl pyrophosphate synthetase 1 (Prps1), and the binding of PPARγ to these motifs was confirmed by using chromatin immunoprecipitation assay. In conclusion, these results demonstrated that TZDs induced alterations in purine nucleotide metabolism in rat hearts via transcriptional regulation of the PPARγ-target genes, which may play an important role in the development of cardiac hypertrophy associated with TZDs.

With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 Å resolution.

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2'-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent Km of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.

A common thread among conserved life span regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of AMP biosynthetic enzymes extend Drosophila life span. The life span benefit of these mutations depends upon increased AMP:ATP and ADP:ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended life span, while AMPK RNAi reduced life span. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed life span extension. Remarkably, this simple change in diet also blocked the prolongevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult life span; provide a mechanistic link between cellular anabolism and energy sensing pathways; and indicate that dietary adenine manipulations might alter metabolism to influence animal life span., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.

In this study, we demonstrate that purA and purB transposon mutants of serotype 4b Listeria monocytogenes were severely impaired in their ability to colonize the gastrointestinal tract and cause systemic infection of the spleen, liver, and gallbladder following intragastric inoculation of A/J mice. The mutant strains were also impaired in their ability to multiply within Caco-2 human intestinal epithelial cells. Neither mutant was affected in resistance to synthetic gastric fluid (pH 4.5). These findings indicate that purine biosynthesis is critical for gastrointestinal virulence of L. monocytogenes serotype 4b in mice.

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp(+) selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade(+) selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5' and 3' homologous sequences, respectively. The resulting Trp(+) transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²+ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K(m) values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k(cat) value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5'-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated. Second, two genes for purine nucleoside phosphorylase, punA and deoD, were inactivated. Third, to enhance purine nucleotide biosynthesis, the pur operon repressor PurR and the 5'-UTR of the operon, containing the guanine riboswitch, were disrupted. Finally, the -10 sequence of the pur promoter was optimized to elevate its transcription level. The resulting mutant was capable of producing 6 g/L inosine from 30 g/L glucose in culture broth without the detectable by-production of hypoxanthine. This indicates the validity of this approach for the breeding of the next generation of B. subtilis strains for industrial nucleoside production.

Tobacco smoke consists of numerous carcinogens whose effect on lung tumor development includes the induction of mutations in key genes as well as the induction of chromosome instability (CIN). Consequently, carcinogen-induced mouse lung adenocarcinomas (LAC) display many more recurrent site- and chromosome-specific changes in DNA copy number compared with noninduced LAC. Here we identified the Adenylosuccinate synthetase 1 (Adss1) gene located on distal chromosome 12q as a focus of bi-allelic or homozygous deletion (HD) in LAC. HDs of Adss1 were detected in 10 out of 84 carcinogen-induced mouse primary LAC and mouse LAC cell lines. In only four of these cases did the deletions affect either Siva1 or Inverted-formin 2 (Inf2), which immediately flank the Adss1 locus, indicating that Adss1 is a selective target of deletion in LAC. Losses of Adss1 not meeting the quantitative threshold of HD were detected in 36 out of 84 (42.9%) of the mouse tumors and cell lines. A similar frequency of ADSS1 deletion was observed in human LAC cell lines, suggesting relevance in human lung cancer. Adss1 losses were also found to be significantly associated with a more extensive CIN phenotype in the primary mouse tumors. These results implicate ADSS1 inactivation as a novel somatic alteration in lung carcinogenesis, and suggest that its selective deletion in LAC may be triggered by CIN.

Background: The blood-derived RNA levels of the adenylosuccinate synthase (ADSS) and ataxia telangiectasia mutated (ATM) genes were found to be down- and up-regulated, respectively, in schizophrenics compared with controls, and ADSS and ATM were among eight biomarker genes to discriminate schizophrenics from normal controls. ADSS catalyzes the first committed step of AMP synthesis, while ATM kinase serves as a key signal transducer in the DNA double-strand breaks response pathway. It remains unclear whether these changes result from mutations or polymorphisms in the two genes., Methods: Six SNPs in the ADSS gene and three SNPs in the ATM gene in a Chinese population of 488 schizophrenics and 516 controls were genotyped to examine their association with schizophrenia (SZ). Genotyping was performed using the Sequenom platform., Results: There was no significant difference in the genotype, allele, or haplotype distributions of the nine SNPs between cases and controls. Using the Multifactor Dimensionality Reduction (MDR) method, we found that the interactions among rs3102460 in the ADSS gene and rs227061 and rs664143 in the ATM gene revealed a significant association with SZ. This model held a maximum testing accuracy of 60.4% and a maximum cross-validation consistency of 10 out of 10., Conclusion: These findings suggest that the combined effects of the polymorphisms in the ADSS and ATM genes may confer susceptibility to the development of SZ in a Chinese population.

Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.

Adenylosuccinate synthetase (ADSS) catalyzes the key step of AMP synthesis. Vertebrates have two isozymes of ADSS, which are named ADSS1 and ADSS2, respectively. In this study, we cloned porcine ADSS1 and ADSS2 genes and comparatively analyzed their sequence, chromosome mapping, mRNA distribution and subcellular localization. According to our results, the ADSS1 gene was predominantly expressed in the striated muscle tissues, while ADSS2 gene distributed widely in all the tissues detected. Additionally, ADSS1 gene was up-regulated significantly along with porcine muscle growth, and ADSS2 gene expression was more constant during the muscle development. Porcine ADSS1 gene was assigned to SSC7q and the linked marker was SSC12B09, ADSS2 gene was mapped on SSC10p and the linked marker was SW497, and porcine ADSS2 protein was subcellular localized in mitochondria. Moreover, we found that one single nucleotide polymorphism (SNP, T/C(70)) in the ninth intron of ADSS2 gene was significantly associated with average daily gain trait (ADG, P<0.05) and loin muscle area trait (P<0.05).

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5' untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.

The Bacillus subtilis purine repressor, PurR, regulates many genes involved in purine metabolism. These genes contain a conserved 14-nucleotide inverted repeat (PurBox). Both pur operon and purA, which are regulated by PurR, have this inverted repeat with a 16- or 17-nucleotide spacer, respectively. Mutational studies have earlier shown that PurR binding is dependent on the PurBox of pur operon. In contrast, these studies failed to establish the importance of purA PurBox to PurR binding. To examine this inconsistency, we studied the effects of PurBox mutations both in vivo and in vitro. The data presented here indicate that purA PurBox has a similar role as pur operon PurBox in PurR binding. In addition, our data suggest that the previously proposed classification of the two halves of the Purbox into weak and strong may need to be revised.

Unlike Borrelia burgdorferi, the relapsing fever agent Borrelia hermsii and the related Borrelia miyamotoi had purA and purB genes of the purine salvage pathway. These were located among the rRNA genes. Phylogenetic analysis indicated that these genes had a different evolutionary history than those of orthologs in other spirochetes.

Aspartate is one of the compounds that induce the differentiation process of the non-infective epimastigote stage to the infective trypomastigote stage of the protozoan parasite Trypanosoma cruzi. l-aspartate is transported by both epimastigote and trypomastigote cells at the same rate, about 3.4 pmolmin(-1) per 10(7) cells. Aspartate transport is only competed by glutamate suggesting that this transport system is specific for anionic amino acids. Aspartate uptake rates increase along the parasite growth curve, by amino acids starvation or pH decrease. The metabolic fate of the transported aspartate was predicted in silico by identification of seven putative genes coding for enzymes involved in aspartate metabolism that could be related to the differentiation process.

Vertebrates have muscle and non-muscle isozymes of adenylosuccinate synthetase (AdSS, EC 6.3.4.4), which catalyzes the first committed step in AMP synthesis. A novel muscle isozyme of adenylosuccinate synthetase, human AdSSL1, is identified from human bone marrow stromal cells. AdSSL1 is 98% identical to mouse muscle type AdSS1 and contains conserved sequence and structural features of adenylosuccinate synthetase. Human AdSSL1 gene is mapped to chromosome 14p32.33. After stimulation, leukemia cells express AdSSL1 in a time-dependent manner different from that of non-muscle adenylosuccinate synthetase. The human AdSSL1 is predominantly expressed in skeletal muscle and cardiac tissue consistent with the potential role for the enzyme in muscle metabolism. Overexpressed AdSSL1 protein in COS-7 cells locates in cytoplasm. Recombinant AdSSL1 protein possesses typical enzymatic activity to catalyze adenylosuccinate formation. The identification of human AdSSL1 with predominant expression in muscle tissue will facilitate future genetic and biochemical analysis of the enzyme in muscle physiology.

Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (psiPAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle.

The specific activities of key enzymes relating to the pathway of inosine synthesis of three different bacterial strains including high-yield, low-yield and wild strains were determined and compared systematically. A close relationship between inosine production and the specific activities of key enzymes was found. According to the enzyme characteristics of high-yield strain, suggestions on further strain improvement by modification of genetic engineering were proposed. Enzymology study is believed to be an effective way to make screening of high-yield strains more efficient.

The addition of glutamine as a major nutrient to cultured neonatal rat cardiomyocytes produced an increase in myocyte size and the organization of actin into myofibrillar arrays. The cellular response was associated with increased abundance of the mRNAs encoding the contractile proteins, alpha-myosin heavy chain and cardiac alpha-actin, and the metabolic enzymes, muscle carnitine palmitoyl transferase I and muscle adenylosuccinate synthetase (ADSS1). Adss1 gene expression was induced approximately 5-fold in glutamine-treated rat neonatal cardiac myocytes. The induction was mediated through the protein kinase A and mammalian target of rapamycin signaling pathways and required a cyclic AMP response element associated with the promoter region of the Adss1 gene. These results highlight glutamine as a major nutrient regulator of cardiac gene expression and identify protein kinase A and mammalian target of rapamycin signaling pathways as mediators of the cardiomyocyte transcriptional response.

Vertebrates have acidic and basic isozymes of adenylosuccinate synthetase, which participate in the first committed step of de novo AMP biosynthesis and/or the purine nucleotide cycle. These isozymes differ in their kinetic properties and N-leader sequences, and their regulation may vary with tissue type. Recombinant acidic and basic synthetases from mouse, in the presence of active site ligands, behave in analytical ultracentrifugation as dimers. Active site ligands enhance thermal stability of both isozymes. Truncated forms of both isozymes retain the kinetic parameters and the oligomerization status of the full-length proteins. AMP potently inhibits the acidic isozyme competitively with respect to IMP. In contrast, AMP weakly inhibits the basic isozyme noncompetitively with respect to all substrates. IMP inhibition of the acidic isozyme is competitive, and that of the basic isozyme noncompetitive, with respect to GTP. Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations. The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme seem functionally independent. Fructose 1,6-bisphosphate and IMP together may be dynamic regulators of the basic isozyme in muscle, causing potent inhibition of the synthetase under conditions of high AMP deaminase activity.

Adenylosuccinate synthetase 1 (ADSS1) functions as an important component in adenine nucleotide biosynthesis and is abundant in the heart. Here we report that the Adss1 gene is up-regulated in two in vivo rodent models of surgically induced cardiac hypertrophy. In addition, we examined an in vitro hypertrophy system of rat neonatal cardiomyocytes treated with angiotensin II to study Adss1 gene regulation. We show that this stimulus triggers a signaling cascade that results in the activation of the Adss1 gene. The induction of Adss1 gene expression was blocked by cyclosporin A in vitro, suggesting that calcineurin, a calmodulin activated phosphatase, is involved in this signaling pathway. Consistent with this view we provide evidence that the induction of Adss1 by angiotension II requires the presence of an NFAT binding site located 556 base pairs upstream of the Adss1 transcription start site. We propose that ADSS1 plays a role in the development of cardiac hypertrophy through its function in adenine nucleotide biosynthesis.

The oxpA5 mutation in Aspergillus nidulans results in a pleiotropic phenotype, including resistance to oxypurinol and partial constitutivity of the enzymes of purine catabolism. Here we show that the oxpA5 mutation is an allele of adB, the gene encoding adenylosuccinate synthetase (ASS). Cloning, sequencing and characterisation of the adB gene are reported in this paper. In vivo complementation tests indicate that the oxpA5 mutation is a partial loss-of-function mutation, and altered kinetic parameters of the ASS could account for the pleiotropic phenotype of the oxpA5 mutant. The transcriptional regulation of adB presents some interesting features, including increased gene expression in the presence of ammonium and of AMP, the final product of purine biosynthesis. The adB gene is located adjacent to helA, a newly identified gene coding for a putative RNA helicase.

For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.

Catalyzing the first step in the de novo synthesis of adenylmonophosphate, adenylosuccinate synthetase (AdSS) is a known target for herbicides and antibiotics. We have purified and crystallized recombinant AdSS from Arabidopsis thaliana and Tritium aestivum, expressed in Escherichia coli. The structures of A. thaliana and T. aestivum AdSS in complex with GDP were solved at 2.9 A and 3.0 A resolution, respectively. Comparison with the known structures from E. coli reveals that the overall fold is very similar to that of the E. coli protein. The longer N terminus in the plant sequences is at the same place as the longer C terminus of the E. coli sequence in the 3D structure. The GDP-binding sites have one additional hydrogen-bonding partner, which is a plausible explanation for the lower K(m) value. Due to its special position, this partner may also enable GTP to initiate a conformational change, which was, in E. coli AdSS, exclusively activated by ligands at the IMP-binding site. The dimer interfaces show up to six hydrogen bonds and six salt-bridges more than in the E. coli structure, although the contact areas have approximately the same size., (Copyright 2000 Academic Press.)

Electrically stimulated pacing of cultured cardiomyocytes serves as an experimentally convenient and physiologically relevant in vitro model of cardiac hypertrophy. Electrical pacing triggers a signaling cascade that results in the activation of the muscle-specific Adss1 gene and the repression of the nonmuscle Adss2 isoform. Activation of the Adss1 gene involves the calcineurin-mediated dephosphorylation of NFAT3, allowing its translocation to the nucleus, where it can directly participate in Adss1 gene activation. Mutational studies show that an NFAT binding site located in the Adss1 5'-flanking region is essential for this activation. Electrical pacing also results in the increased synthesis of GATA4, another critical cardiac transcription factor required for Adss1 gene expression. MEF2C also produces transactivation of the Adss1 gene reporter in control and paced cardiac myocytes. Using the Adss1 gene as a model, these studies are the first to demonstrate that electrical pacing activates the calcineurin/NFAT3 and GATA4 pathways as a means of regulating cardiac gene expression.

Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine. We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S. cerevisiae. The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S. cerevisiae. This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP. l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0. 8-0.9). This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms. Another unusual feature of the enzyme from S. cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.

The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5'-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenous Adss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and these cis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2.