Convergent evolution of nitrogen-adding enzymes in the purine nucleotide biosynthetic pathway, based on structural analysis of adenylosuccinate synthetase (PurA).

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.

ZmAdSS1 encodes adenylosuccinate synthetase and plays a critical role in maize seed development and the accumulation of nutrients.

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.

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA.

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.

A widespread pathway for substitution of adenine by diaminopurine in phage genomes.

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.

A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes.

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.

Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.

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.

ADSSL1 myopathy is the most common nemaline myopathy in Japan with variable clinical features.

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.

Expanding the disease phenotype of ADSSL1-associated myopathy in non-Korean patients.

Adenylosuccinate synthase (ADSSL1) is a muscle specific enzyme involved in the purine nucleotide cycle and responsible for the conversion of inosine monophosphate to adenosine monophosphate. Since 2016, when mutations in the ADSSL1 gene were first described to be associated with an adult onset distal myopathy, nine patients with compound heterozygous variants in the ADSSL1 gene, all of Korean origin, have been identified. Here we report a novel ADSSL1 mutation and describe two sporadic cases of Turkish and Indian origin. Many of the clinical features of both patients and muscle histopathology and muscle MRI findings, were in accordance with previously reported findings in the adult onset distal myopathy individuals. However, one of our patients presented with progressive, proximally pronounced weakness, severe muscle atrophy and early contractures. Thus, mutations in ADSSL1 have to be considered in patients with both distal and proximal muscle weakness and across various ethnicities.

Increased fermentative adenosine production by gene-targeted Bacillus subtilis mutation.

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.

Identification of Staphylococcus aureus Factors Required for Pathogenicity and Growth in Human Blood.

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.

Distal myopathy with ADSSL1 mutations in Korean patients.

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.

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.

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.

Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

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.

ADSSL1 mutation relevant to autosomal recessive adolescent onset distal myopathy.

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.

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet ß 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 ß 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 ß cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing ß cell dysfunction in T2D.

Increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya gossypii.

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.

Metabonomic profiling revealed an alteration in purine nucleotide metabolism associated with cardiac hypertrophy in rats treated with thiazolidinediones.

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.

Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans.

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 Šresolution.

Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

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 Šresolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, ß = 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%.

Adenylosuccinate synthetase and adenylosuccinate lyase deficiencies trigger growth and infectivity deficits in Leishmania donovani.

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.