Conserved bacterial de novo guanine biosynthesis pathway enables microbial survival and colonization in the environmental niche of the urinary tract.
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ABSTRACT:
SUBMITTER: Ipe DS
PROVIDER: S-EPMC8245529 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical properties. Early literature evidence suggested that they might assemble into a multi-enzyme complex called a metabolon. The finding that fluorescently-tagged chimeras of the pathway enzymes form discrete puncta, now called purinosomes, is further elaborated in this review to include: a discussion of their assembly; the role of ancillary proteins; their locus at the microtubule/mitochondria interface; the elucidation that at endogenous levels, purinosomes function to channel intermediates from phosphoribosyl pyrophosphate to AMP and GMP; and the evidence for the purinosomes to exist as a protein condensate. The review concludes with a consideration of probable signaling pathways that might promote the assembly and disassembly of the purinosome, in particular the identification of candidate kinases given the extensive phosphorylation of the enzymes. These collective findings substantiate our current view of the de novo purine biosynthetic metabolon whose properties will be representative of how other metabolic pathways might be organized for their function.
| S-EPMC7869020 | biostudies-literature
Project description:The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.
| S-EPMC17790 | biostudies-literature
Project description:UnlabelledDe novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility.ImportanceDe novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.
| S-EPMC4936099 | biostudies-literature
Project description:The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes. Here, we show the discovery and characterization of these two elusive reductases, alongside the identification of two physiologically relevant esterases that complete the biosynthesis of ajmaline. We show that ajmaline biosynthesis proceeds with vomilenine 1,2(R)-reduction followed by its 19,20(S)-reduction. This process is further modulated by two root-expressing esterases that deacetylate 17-O-acetylnorajmaline. Expanding upon the successful completion of the ajmaline biosynthetic pathway, we engineer the de novo biosynthesis of ajmaline in Baker's yeast.
| S-EPMC10784492 | biostudies-literature
Project description:Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.
| S-EPMC1152552 | biostudies-other
Project description:Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to "non-natural" product target compounds.
| S-EPMC6001691 | biostudies-literature
Project description:Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.
| S-EPMC4296673 | biostudies-literature
Project description:Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.
| S-EPMC6287904 | biostudies-literature
Project description:Mycobacteria can synthesize NAD+ using either the de novo biosynthesis pathway or the salvage pathway. The deletion of the three genes involved specifically in the NAD+ de novo biosynthesis pathway in the human pathogen Mycobacterium tuberculosis had no effect on the growth of the strain in vivo. In contrast, the same deletion in the bovine pathogen Mycobacterium bovis resulted in a strain that could not grow in vivo and could only grow in vitro with substantial nicotinamide supplmentation. This striking difference was attributed to the known defect in the nicotinamidase PncA of M. bovis, since introducing the M. tuberculosis pncA gene into the M. bovis strain defective for de novo NAD+ biosynthesis restored growth in vitro and in vivo. This study demonstrates that NAD+ starvation is a cidal event in mycobacteria and confirms that enzymes common to the de novo and salvage pathways may be good drug targets. We also propose that simultaneously targeting both the salvage and the de novo NAD+ biosynthesis pathways represents a potentially effective way to treat infection with tubercle bacilli. To characterize the lethality induced by nicotinamide starvation transcriptional profiling of the auxotrophs was performed. Triplicate 50 mL cultures of M. tuberculosis and M. bovis Delta nadABC mutants were grown in 7H9 OADC glycerol 0.05% tween broth in 500 mL roller bottles to an OD600nm= 0.1 in a roller incubator at 37°C. The cells were washed 1x in PBS and resuspended in 50 mL 7H9 OADC glycerol 0.05% tween broth with or without 20mg/L nicotinamide and returned to the incubator. After 7 days, cultures were harvested. Three biological replicates of each of two species with one dye-flip each
2010-05-14 | E-GEOD-18909 | biostudies-arrayexpress
Project description:AimTo test the effects of dapagliflozin-induced hyperglucosuria on ascending bacterial urinary tract infection (UTI) in a mouse model.MethodsDapagliflozin or canagliflozin was used to induce hyperglucosuria in non-diabetic adult female mice prior to transurethral inoculation with uropathogenic Escherichia coli (UPEC) or Klebsiella pneumoniae. Glucose, bacterial load, cytokines, neutrophil mobilization and inflammation during acute and chronic UTI were determined.ResultsSignificant increase in UPEC load was observed in the urinary tract of hyperglucosuric mice compared with controls. Dapagliflozin-treated mice developed bacteraemia resulting in UPEC colonization of the spleen and liver at a higher frequency than controls. Chronic UTI in hyperglucosuric mice resulted in an increased incidence of renal abscesses. Histopathological evaluation revealed only modest increases in tissue damage in the urinary bladders and kidneys of dapagliflozin-treated mice, despite a profound increase in bacterial load. There was poor neutrophil mobilization to the urine of hyperglucosuric mice. We also observed a delayed increase of IL-1β in urine, and bladders, and IL-6 in urine of hyperglucosuric mice. Experimental inoculation with K. pneumoniae also revealed higher bacterial burden in the urinary bladder, spleen and liver from dapagliflozin-treated mice compared with controls.ConclusionCollectively, our results indicate that dapagliflozin-induced hyperglucosuria in non-diabetic female mice leads to increased susceptibility to severe UTI, and bacteraemia of urinary tract origin.
| S-EPMC7571118 | biostudies-literature