RESUMEN
In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-ß-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.
Asunto(s)
Mycobacterium tuberculosis , Fosfatos de Poliisoprenilo , Mycobacterium tuberculosis/genética , Fosforribosil Pirofosfato/metabolismo , Antituberculosos/metabolismo , Pared Celular/metabolismoRESUMEN
The pentose phosphate pathway (PPP) is critical for anabolism and biomass production. Here we show that the essential function of PPP in yeast is the synthesis of phosphoribosyl pyrophosphate (PRPP) catalyzed by PRPP-synthetase. Using combinations of yeast mutants, we found that a mildly decreased synthesis of PRPP affects biomass production, resulting in reduced cell size, while a more severe decrease ends up affecting yeast doubling time. We establish that it is PRPP itself that is limiting in invalid PRPP-synthetase mutants and that the resulting metabolic and growth defect can be bypassed by proper supplementation of the medium with ribose-containing precursors or by the expression of bacterial or human PRPP-synthetase. In addition, using documented pathologic human hyperactive forms of PRPP-synthetase, we show that intracellular PRPP as well as its derived products can be increased in both human and yeast cells, and we describe the ensuing metabolic and physiological consequences. Finally, we found that PRPP consumption appears to take place "on demand" by the various PRPP-utilizing pathways, as shown by blocking or increasing the flux in specific PRPP-consuming metabolic routes. Overall, our work reveals important similarities between human and yeast for both synthesis and consumption of PRPP.
Asunto(s)
Fosforribosil Pirofosfato , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Bacterias , Vía de Pentosa Fosfato , LigasasRESUMEN
To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.
Asunto(s)
Cobre , Staphylococcus aureus , Animales , Ratones , Staphylococcus aureus/metabolismo , Vía de Pentosa Fosfato , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Fosforribosil Pirofosfato/metabolismo , AdeninaRESUMEN
Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors - NAD and NADP, the amino acids histidine and tryptophan biosynthesis. We aimed to investigate the impact of the different effectors on the activity of PRPS-1, to check the activity of the enzyme in vitro in a wide range of pHs and investigate some structural essentials of the enzyme, isolated from brain and liver. Molecular docking analyses were used to delineate the essentials of PRPS-1 structure, to find out the existence of enzyme effectors. Previously created by us kit was used for determination of the activity of PRPS-1 based on the formation of the inorganic phosphates (λ = 700 nm, Cary 60, Agilent, USA). Effectors impact on the activity of PRPS-1 was evaluated. In silico results of the effectors were later proven by in vitro experiments. For the first time biochemical essentials, including the existence of the multiple pockets, involvement of the amino acids into the processes of interactions with the effectors, evolutional of the sequence conservation, tissue depended Vmax differences were identified.
Asunto(s)
Fosforribosil Pirofosfato , Ribosa-Fosfato Pirofosfoquinasa , Difosfatos , Histidina , Simulación del Acoplamiento Molecular , NAD , NADP , Nucleótidos , Fosfatos , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Purinas/metabolismo , Piridinas , Pirimidinas , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , TriptófanoRESUMEN
Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD+) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD+ during DNA repair. Mammalian cells produce NAD+ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD+ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD+ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD+ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.
Asunto(s)
Proteínas Quinasas Activadas por AMP , NAD , Nicotinamida Fosforribosiltransferasa , Radiación Ionizante , Proteínas Quinasas Activadas por AMP/metabolismo , Citocinas/metabolismo , Homeostasis , Humanos , NAD/metabolismo , Niacinamida , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Fosforribosil PirofosfatoRESUMEN
Phosphoribosyl pyrophosphate synthetase (PRS EC 2.7.6.1) is a rate-limiting enzyme that irreversibly catalyzes the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate (ATP). This key metabolite is required for the synthesis of purine and pyrimidine nucleotides, the two aromatic amino acids histidine and tryptophan, the cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), all of which are essential for various life processes. Despite its ubiquity and essential nature across the plant and animal kingdoms, PRPP synthetase displays species-specific characteristics regarding the number of gene copies and architecture permitting interaction with other areas of cellular metabolism. The impact of mutated PRS genes in the model eukaryote Saccharomyces cerevisiae on cell signalling and metabolism may be relevant to the human neuropathies associated with PRPS mutations. Human PRPS1 and PRPS2 gene products are implicated in drug resistance associated with recurrent acute lymphoblastic leukaemia and progression of colorectal cancer and hepatocellular carcinoma. The investigation of PRPP metabolism in accepted model organisms, e.g., yeast and zebrafish, has the potential to reveal novel drug targets for treating at least some of the diseases, often characterized by overlapping symptoms, such as Arts syndrome and respiratory infections, and uncover the significance and relevance of human PRPS in disease diagnosis, management, and treatment.
Asunto(s)
Fosforribosil Pirofosfato , Pez Cebra , Adenosina Trifosfato/química , Animales , Humanos , NAD , Recurrencia Local de Neoplasia , Fosforribosil Pirofosfato/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Pez Cebra/metabolismoRESUMEN
Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.
Asunto(s)
Escherichia coli , Fosforribosil Pirofosfato , Regulación Alostérica , Sitio Alostérico , Escherichia coli/genética , Humanos , Fosforribosil Pirofosfato/químicaRESUMEN
Phosphoribosyl pyrophosphate synthetases (EC 2.7.6.1) are key enzymes in the biological synthesis of phosphoribosyl pyrophosphate and are involved in diverse developmental processes. In our previous study, the PRPS1 gene was discovered as a key disease-resistance candidate gene in yellow drum, Nibea albiflora, in response to the infection of Vibrio harveyi, through genome-wide association analysis. This study mainly focused on the characteristics and its roles in immune responses of the PRPS1 gene in yellow drum. In the present study, the NaPRPS1 gene was cloned from yellow drum, encoding a protein of 320 amino acids. Bioinformatic analysis showed that NaPRPS1 was highly conserved during evolution. Quantitative RT-PCR demonstrated that NaPRPS1 was highly expressed in the head-kidney and brain, and its transcription and translation were significantly activated by V. harveyi infection examined by RT-qPCR and immunohistochemistry analysis, respectively. Subcellular localization revealed that NaPRPS1 was localized in cytoplasm. In addition, semi-in vivo pull-down assay coupled with mass spectrometry identified myeloid differentiation factor 88 (MyD88) as an NaPRPS1-interacting patterner, and their interaction was further supported by reciprocal pull-down assay and co-immunoprecipitation. The inducible expression of MyD88 by V. harveyi suggested that the linker molecule MyD88 in innate immune response may play together with NaPRPS1 to coordinate the immune signaling in yellow drum in response to the pathogenic infection. We provide new insights into important functions of PRPS1, especially PRPS1 in the innate immunity of teleost fishes, which will benefit the development of marine fish aquaculture.
Asunto(s)
Enfermedades de los Peces , Perciformes , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Vibrio , Animales , Enfermedades de los Peces/genética , Peces/genética , Estudio de Asociación del Genoma Completo , Inmunidad Innata/genética , Factor 88 de Diferenciación Mieloide/genética , Perciformes/genética , Fosforribosil Pirofosfato , Vibrio/fisiologíaRESUMEN
Cells coordinate their behaviors with the mechanical properties of the extracellular matrix (ECM). Tumor cells frequently harbor an enhanced nucleotide synthesis, presumably to meet the increased demands for rapid proliferation. Nevertheless, how ECM rigidity regulates nucleotide metabolism remains elusive. Here we show that shift from stiff to soft matrix blunts glycolysis-derived nucleotide synthesis in tumor cells. Soft ECM results in TNF receptor-associated factor 2 (TRAF2)-dependent K29 ubiquitination and degradation of phosphoribosyl pyrophosphate synthetase (PRPS)1/2. Recruitment of TRAF2 to PRPS1/2 requires phosphorylation of PRPS1 S285 or PRPS2 T285, which is mediated by low stiffness-activated large tumor suppressor (LATS)1/2 kinases. Further, non-phosphoryable or non-ubiquitinatable PRPS1/2 mutations maintain PRPS1/2 expression and nucleotide synthesis at low stiffness, and promote tumor growth and metastasis. Our findings demonstrate that PRPS1/2 stability and nucleotide metabolism is ECM rigidity-sensitive, and thereby highlight a regulatory cascade underlying mechanics-guided tumor metabolism reprogramming.
Asunto(s)
Fosforribosil Pirofosfato , Ribosa-Fosfato Pirofosfoquinasa , Ligasas/metabolismo , Nucleótidos/metabolismo , Fosforilación , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.
Asunto(s)
Antimetabolitos/farmacología , Antivirales/farmacología , Virus ARN/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Amidas/farmacología , Animales , Línea Celular , Sinergismo Farmacológico , Guanosina Trifosfato/metabolismo , Humanos , Metiltioinosina/farmacología , Mutación/efectos de los fármacos , Fosforribosil Pirofosfato/metabolismo , Pirazinas/farmacología , Virus ARN/clasificación , Virus ARN/genética , ARN Viral/efectos de los fármacos , ARN Viral/genética , Replicación Viral/efectos de los fármacosRESUMEN
Ionizing radiation-induced DNA damages cause genome instability and are highly cytotoxic. Deoxyribonucleotide metabolism provides building blocks for DNA repair. Nevertheless, how deoxyribonucleotide metabolism is timely regulated to coordinate with DNA repair remains elusive. Here, we show that ionizing radiation results in TBK1-mediated phosphorylation of phosphoribosyl pyrophosphate synthetase (PRPS)1/2 at T228, thereby enhancing PRPS1/2 catalytic activity and promoting deoxyribonucleotide synthesis. DNA damage-elicited activation of cGAS/STING axis and ATM-mediated PRPS1/2 S16 phosphorylation are required for PRPS1/2 T228 phosphorylation under ionizing radiation. Furthermore, T228 phosphorylation overrides allosteric regulator-mediated effects and preserves PRPS1/2 with high activity. The expression of non-phosphorylatable PRPS1/2 mutants or inhibition of cGAS/STING axis counteracts ionizing radiation-induced PRPS1/2 activation, deoxyribonucleotide synthesis, and DNA repair, and further impairs cell viability. This study highlights a novel and important mechanism underlying an innate immune response-guided deoxyribonucleotide metabolism, which supports DNA repair.
Asunto(s)
Nucleotidiltransferasas , Fosforribosil Pirofosfato , Reparación del ADN , Inmunidad Innata , Ligasas/metabolismo , Nucleotidiltransferasas/metabolismo , FosforilaciónRESUMEN
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.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteínas/metabolismo , Purinas/biosíntesis , Transducción de Señal/fisiología , Adenosina Monofosfato/metabolismo , Vías Biosintéticas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Guanosina Monofosfato/metabolismo , Humanos , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Fosforribosil Pirofosfato/metabolismo , FosforilaciónRESUMEN
Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin's effects. Amino acid deprivation enhanced cisplatin's cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin's biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug's side effects, and allow its use in cisplatin-resistant tumors.
Asunto(s)
Aminoácidos/deficiencia , Apoptosis , Carcinoma Hepatocelular/patología , Cisplatino/farmacología , Neoplasias Hepáticas/patología , Fosforribosil Pirofosfato/metabolismo , Animales , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The enzyme ForT catalyzes C-C bond formation between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5'-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.
Asunto(s)
Ligasas de Carbono-Carbono/metabolismo , Fosforribosil Pirofosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Ligasas de Carbono-Carbono/química , Dominio Catalítico , Cristalografía por Rayos X , Modelos Químicos , Fosforribosil Pirofosfato/química , Unión Proteica , Streptomyces/enzimologíaRESUMEN
l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Histidina/biosíntesis , Ingeniería Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Corynebacterium glutamicum/metabolismo , Fermentación , Glutamato Deshidrogenasa/metabolismo , Lisina/metabolismo , Fosforribosil Pirofosfato/metabolismo , Nucleótidos de Purina/biosíntesisRESUMEN
The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.
Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/genética , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/metabolismo , Ligandos , Modelos Moleculares , Conformación de Ácido Nucleico , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismoRESUMEN
Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.
Asunto(s)
Fosforribosil Pirofosfato/biosíntesis , Proteínas Recombinantes de Fusión , Cromatografía de Afinidad , Clonación Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , TemperaturaRESUMEN
The five-membered PRS gene family of Saccharomyces cerevisiae is an example of gene duplication allowing the acquisition of novel functions. Each of the five Prs polypeptides is theoretically capable of synthesising PRPP but at least one of the following heterodimers is required for survival: Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5. Prs3 contains a pentameric motif 284KKCPK288 found only in nuclear proteins. Deletion of 284KKCPK288 destabilises the Prs1/Prs3 complex resulting in a cascade of events, including reduction in PRPP synthetase activity and altered cell wall integrity (CWI) as measured by caffeine sensitivity and Rlm1 expression. Prs3 also interacts with the kinetochore-associated protein, Nuf2. Following the possibility of 284KKCPK288-mediated transport of the Prs1/Prs3 complex to the nucleus, it may interact with Nuf2 and phosphorylated Slt2 permitting activation of Rlm1. This scenario explains the breakdown of CWI encountered in mutants lacking PRS3 or deleted for 284KKCPK288. However, removal of NHR1-1 from Prs1 does not disrupt the Prs1/Prs3 interaction as shown by increased PRPP synthetase activity. This is evidence for the separation of the two metabolic functions of the PRPP-synthesising machinery: provision of PRPP and maintenance of CWI and is an example of evolutionary development when multiple copies of a gene were present in the ancestral organism.
Asunto(s)
Secuencias de Aminoácidos , Viabilidad Microbiana , Fosforribosil Pirofosfato/biosíntesis , Subunidades de Proteína/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Unión Proteica , Mapas de Interacción de Proteínas , Subunidades de Proteína/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrolasas/metabolismo , Fosforribosil Pirofosfato/metabolismo , ARN Bacteriano/metabolismo , Riboswitch/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Enterobacteriaceae/metabolismo , Hidrolasas/genética , Leuconostoc mesenteroides/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/genéticaRESUMEN
Relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5-Fluorouracil (5-FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5-FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5-FU to FUMP and FUTP in Reh cells, to promote 5-FU-induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re-targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse-specific mutations of PRPS1 might benefit from 5-FU-based chemotherapy in clinical settings.