Insights into the ATP / GTP selectivity of a GTPase, adenylosuccinate synthetase from Leishmania donovani.

Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 (593SAXD596) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity.

Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli.

Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.

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.

High fructose-induced skeletal muscle insulin resistance could be alleviated by berberine via AMPD1 and ADSL.

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis that is activated in response to an elevated intracellular AMP/ATP ratio. Although many studies have shown berberine is an AMPK activator widely used in metabolic syndrome, how to properly control AMPK activity remains obscure. Our present study aimed to examine the protective effect of berberine against fructose-induced insulin resistance in rats and L6 cells, as well as its potential activation mechanism on AMPK. The results showed that berberine effectively reversed body weight gain, Lee's index, dyslipidemia and insulin intolerance. Moreover, berberine alleviated inflammatory response, antioxidant capacity and promoted glucose uptake in vivo and in vitro. The beneficial effect was associated with upregulation of both Nrf2 and AKT/GLUT4 pathways, which were regulated by AMPK. Notably, berberine could increase the level of AMP and the ratio of AMP/ATP, then further activate AMPK. Mechanistic experiments revealed that berberine suppressed the expression of adenosine monophosphate deaminase 1 (AMPD1) and promoted the expression of adenylosuccinate synthetase (ADSL). Taken together, berberine exerted excellent therapeutic effect on insulin resistance. And its mode of action may be related to the AMP-AMPK pathway by regulating AMPD1 and ADSL.

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.

The pursuit of new alternative ways to eradicate Helicobacter pylori continues: Detailed characterization of interactions in the adenylosuccinate synthetase active site.

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287-293 and 40-44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40-50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori.

Metformin alleviates long-term high-fructose diet-induced skeletal muscle insulin resistance in rats by regulating purine nucleotide cycle.

Nutrient excess caused by excessive fructose intake can lead to insulin resistance and dyslipidemia, which further causes the development of metabolic syndrome. Metformin is a well-known AMPK activator widely used for the treatment of metabolic syndrome, while the mechanism of AMPK activation remains unclear. The present study aimed to investigate the pharmacological effects of metformin on fructose-induced insulin resistance rat, and the potential mechanism underlying AMPK activation in skeletal muscle tissue. Results indicated that metformin significantly ameliorated features of insulin resistance, including body weight, Lee's index, hyperinsulinemia, dyslipidemia, insulin intolerance and pancreatic damage. Moreover, treatment with metformin attenuated the inflammatory response in serum and enhanced the antioxidant capacity in skeletal muscle tissue. The therapeutic effects of metformin on fructose-induced insulin resistance may be related to the activation of AMPK to regulate Nrf2 pathway and mitochondrial abnormality. Additionally, metformin suppressed the expression of adenosine monophosphate deaminase 1 (AMPD1) and up-regulated the expression of adenylosuccinate synthetase (ADSS) in the purine nucleotide cycle (PNC), which facilitated the increase of AMP level and the ratio of AMP/ATP. Therefore, we proposed a novel mechanism that metformin activated AMPK via increasing AMP by regulating the expression of AMPD1 and ADSS in PNC pathway.

An autopsied case of ADSSL1 myopathy.

ADSSL1 myopathy is an inherited myopathy with limb weakness, respiratory muscle paralysis, dysphagia, and myocardial symptoms. We present an autopsy case of a 66-year-old male carrying compound heterozygous variants c.781G>A (p.D261N) and c.919delA (p.I307fs) in ADSSL1. He had not run fast since school with no family history. He showed a gradual progression of limb weakness and developed dyspnoea, dysphagia, and Brugada syndrome at the age of 56. The magnetic resonance imaging (MRI) revealed bright tongue sign. Muscle biopsy showed only chronic myopathic changes. He died of respiratory muscle weakness at the age of 66. Autopsy revealed that there were many fibres with vacuoles and nemaline rods in the biceps brachii, tongue, diaphragm, and iliopsoas. Many lipopigments and nuclear clumps were also detected. The myocardium and central nervous system had only nonspecific age-related changes. This is the first autopsied case to clarify the terminal state of ADSSL1 myopathy.

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

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.

Comparative transcriptome analysis of skeletal muscle in ADSSL1 myopathy.

ADSSL1 myopathy was recently identified as the cause of muscular disorders in Korean patients with distal myopathy. We generated transcriptome profiles of muscles from control subjects and patients with ADSSL1 myopathy. In the present study, RNA sequencing was conducted with seven vastus lateralis muscle samples from four patients with ADSSL1 myopathy and three control subjects. The hierarchical clustering result revealed a separation between myopathy and control groups. A total of 1,260 transcripts were significantly differentially expressed (|fold change| ≥ 2, p < 0.05), with 740 upregulated transcripts and 520 downregulated transcripts in myopathy group. Eighteen transcripts that mapped to purine metabolism pathway were significantly differentially expressed between the two groups, with ten downregulated transcripts and eight upregulated transcripts in myopathy group. In particular, three genes involved in purine nucleotide cycle (ADSSL1, ADSL, and AMPD1) were significantly downregulated in myopathy group. Ten transcripts in glycolysis/gluconeogenesis pathway were also significantly differentially expressed. This is the first study on the altered expression of transcripts in muscle tissues from patients with ADSSL1 myopathy. Our results provide new insights into the pathogenesis of ADSSL1 myopathy.

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori.

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium's high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

Insight into cordycepin biosynthesis of Cordyceps militaris: Comparison between a liquid surface culture and a submerged culture through transcriptomic analysis.

Cordyceps militaris produces cordycepin, which is known to be a bioactive compound. Currently, cordycepin hyperproduction of C. militaris was carried out in a liquid surface culture because of its low productivity in a submerged culture, however the reason was not known. In this study, 4.92 g/L of cordycepin was produced at the 15th day of C. militaris NBRC 103752 liquid surface culture, but only 1 mg/L was produced in the submerged culture. RNA-Seq was used to clarify the gene expression profiles of the cordycepin biosynthetic pathways of the submerged culture and the liquid surface culture. From this analysis, 1036 genes were shown to be upregulated and 557 genes were downregulated in the liquid surface culture compared with the submerged culture. Specifically, adenylosuccinate synthetase and phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthase in purine nucleotide metabolism were significantly upregulated in the liquid surface culture. Thick mycelia formation in the liquid surface culture was found to induce the expression of hypoxia-related genes (GABA shunt, glutamate synthetase precursor, and succinate-semialdehyde dehydrogenase). Cytochrome P450 oxidoreductases containing heme were also found to be significantly enriched, suggesting that a hypoxic condition might be created in the liquid surface culture. These results suggest that hypoxic conditions are more suitable for cordycepin production in the liquid surface culture compared with the submerged culture. Our analysis paves the way for unraveling the cordycepin biosynthesis pathway and for improving cordycepin production in C. militaris.

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.