Purine biosynthesis Dotaz Zobrazit nápovědu
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.
- MeSH
- chromatografie kapalinová MeSH
- CRISPR-Cas systémy MeSH
- DNA biosyntéza chemie MeSH
- editace genu MeSH
- HeLa buňky MeSH
- lidé MeSH
- puriny biosyntéza chemie MeSH
- RNA biosyntéza chemie MeSH
- tandemová hmotnostní spektrometrie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Folate deficiency in the critical developmental period has been repeatedly associated with an increased risk of Autism spectrum disorders (ASD), but the key pathophysiological mechanism has not yet been identified. In this work, we focused on identifying genes whose defect has similar consequences to folate depletion in the metabolic network. Within the Flux Balance Analysis (FBA) framework, we developed a method of blocked metabolites that allowed us to define the metabolic consequences of various gene defects and folate depletion. We identified six genes (GART, PFAS, PPAT, PAICS, ATIC, and ADSL) whose blocking results in nearly the same effect in the metabolic network as folate depletion. All of these genes form the purine biosynthetic pathway. We found that, just like folate depletion, the blockade of any of the six genes mentioned above results in a blockage of purine metabolism. We hypothesize that this can lead to decreased adenosine triphosphate (ATP) and subsequently, an S-adenosyl methionine (SAM) pool in neurons in the case of rapid cell division. Based on our results, we consider the methylation defect to be a potential cause of ASD, due to the depletion of purine, and consequently S-adenosyl methionine (SAM), biosynthesis.
- Publikační typ
- časopisecké články MeSH
Metabolism of purine bases remains poorly understood in the pathogenic bacterium Mycobacterium tuberculosis and closely related, nonpathogenic Mycobacterium smegmatis (Msm). To gain insight into the purine metabolism in mycobacteria, we tested uptake of purine bases with a ΔpurF Msm mutant with an inactive purine de novo biosynthesis pathway and confirmed that hypoxanthine and guanine, but not xanthine, can serve as nucleotide precursors for recycling in the salvage pathway. Further, we focused on purine catabolism in wild-type (wt) Msm. We found that only xanthine and guanine could serve as a sole nitrogen source for wt Msm. These data confirm that Msm catabolism of purines is directed mainly via oxidative guanine to xanthine interconversion and not through metabolic conversion of hypoxanthine to xanthine. Our data represent the first experimental evidence confirming the use of 8-oxo-purines as a nitrogen source by Msm.
- MeSH
- atypické mykobakteriální infekce metabolismus mikrobiologie MeSH
- guanin metabolismus MeSH
- lidé MeSH
- Mycobacterium smegmatis izolace a purifikace metabolismus MeSH
- puriny metabolismus MeSH
- xanthin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- adenin biosyntéza MeSH
- buněčné jádro enzymologie MeSH
- cytosin biosyntéza MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- DNA biosyntéza MeSH
- formiáty metabolismus MeSH
- krysa rodu rattus MeSH
- kultivační techniky MeSH
- Parvoviridae růst a vývoj MeSH
- RNA biosyntéza MeSH
- thymin biosyntéza MeSH
- uracil biosyntéza MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
