RESUMEN
AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.
Asunto(s)
Adenina Fosforribosiltransferasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Ribonucleótidos/biosíntesis , Acinetobacter/enzimología , Aminoimidazol Carboxamida/química , Bacterias/enzimología , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Polifosfatos/química , Ribonucleótidos/química , TemperaturaRESUMEN
Viperin is an interferon-induced cellular protein that is conserved in animals1. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.
Asunto(s)
Antivirales/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago T7/inmunología , Evolución Molecular , Células Procariotas/metabolismo , Proteínas/metabolismo , Antivirales/inmunología , Proteínas Arqueales/química , Bacterias/inmunología , Bacterias/metabolismo , Bacterias/virología , Proteínas Bacterianas/química , Bacteriófago T7/enzimología , Bacteriófago T7/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Células Procariotas/inmunología , Células Procariotas/virología , Proteínas/química , Proteínas/genética , Ribonucleótidos/biosíntesis , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Prebiotic chemistry aims to explain how the biochemistry of life as we know it came to be. Most efforts in this area have focused on provisioning compounds of importance to life by multistep synthetic routes that do not resemble biochemistry. However, gaining insight into why core metabolism uses the molecules, reactions, pathways, and overall organization that it does requires us to consider molecules not only as synthetic end goals. Equally important are the dynamic processes that build them up and break them down. This perspective has led many researchers to the hypothesis that the first stage of the origin of life began with the onset of a primitive nonenzymatic version of metabolism, initially catalyzed by naturally occurring minerals and metal ions. This view of life's origins has come to be known as "metabolism first". Continuity with modern metabolism would require a primitive version of metabolism to build and break down ketoacids, sugars, amino acids, and ribonucleotides in much the same way as the pathways that do it today. This review discusses metabolic pathways of relevance to the origin of life in a manner accessible to chemists, and summarizes experiments suggesting several pathways might have their roots in prebiotic chemistry. Finally, key remaining milestones for the protometabolic hypothesis are highlighted.
Asunto(s)
Aminoácidos/metabolismo , Origen de la Vida , Ribonucleótidos/metabolismo , Azúcares/metabolismo , Aminoácidos/biosíntesis , Aminoácidos/genética , Metabolismo de los Hidratos de Carbono , Código Genético , Redes y Vías Metabólicas , Ribonucleótidos/biosíntesisRESUMEN
Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Ribonucleótidos/biosíntesis , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Descubrimiento de Drogas/métodos , Genoma BacterianoRESUMEN
AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an adenosine analog, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as minimal essential medium α (MEMα), contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake, and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B-12, vitamin C, and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Metabolismo Energético/genética , Neoplasias/genética , Proteínas Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenosina/genética , Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Ácido Ascórbico/química , Ácido Ascórbico/farmacología , Línea Celular Tumoral , Medios de Cultivo/química , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Glucosa/metabolismo , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Neoplasias/metabolismo , Neoplasias/patología , Nucleósidos/biosíntesis , Nucleósidos/genética , Proteínas Quinasas/metabolismo , Ribonucleótidos/biosíntesis , Ribonucleótidos/genética , Ácido Tióctico/química , Ácido Tióctico/farmacología , Vitamina B 12/química , Vitamina B 12/farmacologíaRESUMEN
Similar to other (+)RNA viruses, tomato bushy stunt virus (TBSV) utilizes metabolites, lipids, membranes, and co-opted host factors during replication. The coordination of cell metabolism and growth with environmental cues is performed by the target of rapamycin (TOR) kinase in eukaryotic cells. In this paper, we find that TBSV replication partially inhibits TOR activity, likely due to recruitment of glycolytic enzymes to the viral replication compartment, which results in reduced ATP levels in the cytosol. Complete inhibition of TOR activity with rapamycin in yeast or AZD8055 inhibitor in plants reduces tombusvirus replication. We find that high glucose concentration, which stimulates TOR activity, enhanced tombusvirus replication in yeast. Depletion of yeast Sch9 or plant S6K1 kinase, a downstream effector of TOR, also inhibited tombusvirus replication in yeast and plant or the assembly of the viral replicase in vitro. Altogether, the TOR pathway is crucial for TBSV to replicate efficiently in hosts.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Nicotiana/virología , ARN Viral/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virología , Tombusvirus/genética , Factores de Transcripción/metabolismo , Replicación Viral , Adenosina Trifosfato/metabolismo , Glucólisis , Interacciones Huésped-Patógeno , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Morfolinas/farmacología , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleótidos/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Sirolimus/farmacología , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , Tombusvirus/fisiología , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5'-(G/C)GGA-3' template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5'-GA-3') as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , ADN Helicasas/metabolismo , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas Multifuncionales/metabolismo , ARN/biosíntesis , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Secuencia Conservada , ADN Helicasas/química , ADN Helicasas/genética , ADN Primasa/química , ADN Primasa/genética , ADN de Cadena Simple/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Enzimas Multifuncionales/química , Enzimas Multifuncionales/genética , Unión Proteica , Ribonucleótidos/biosíntesis , Moldes GenéticosRESUMEN
While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nucleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Fibras Musculares Esqueléticas/enzimología , Ubiquitina-Proteína Ligasas/biosíntesis , Aminoimidazol Carboxamida/análogos & derivados , Animales , Línea Celular , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/metabolismo , Ratas , Ribonucleótidos/biosíntesis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , IMP Deshidrogenasa/metabolismo , Inmunosupresores/uso terapéutico , Leucocitos Mononucleares/enzimología , Ácido Micofenólico/análogos & derivados , Enfermedad Aguda , Adulto , Anciano , Biomarcadores/metabolismo , Femenino , Supervivencia de Injerto , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/mortalidad , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Inosina Monofosfato/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Ácido Micofenólico/uso terapéutico , Pronóstico , Estudios Prospectivos , Recurrencia , Ribonucleótidos/antagonistas & inhibidores , Ribonucleótidos/biosíntesis , Análisis de Supervivencia , Quimera por Trasplante/genética , Quimera por Trasplante/inmunología , Trasplante Homólogo , XantinaRESUMEN
N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Ligasas/genética , Ribonucleótidos/biosíntesis , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bicarbonatos/metabolismo , Sitios de Unión/genética , Catálisis , Dominio Catalítico/genética , Escherichia coli/enzimología , Cinética , Ligasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-DirigidaRESUMEN
High-resolution investigations of the structure and dynamics of RNA molecules by nuclear magnetic resonance (NMR) methodologies require the production of (13)C/(15)N-isotopically labeled samples. A common strategy entails the preparation of (13)C/(15)N-enriched ribonucleoside 5'-triphosphates (rNTPs) to be incorporated into RNA oligomers by in vitro transcription. Here, we describe the methods to obtain isotopically labeled rNTP in a uniform or selective fashion from bacterial cultures, using common and versatile E. coli strains. This chapter also covers procedures for extraction and digestion of the total RNA from bacterial cells, purification of the ribonucleoside 5'-monophosphates and their enzymatic phosphorylation to rNTPs.
Asunto(s)
ARN/biosíntesis , ARN/química , Ribonucleótidos/biosíntesis , Ribonucleótidos/química , Acetatos/farmacología , Isótopos de Carbono/química , Proliferación Celular , Medios de Cultivo/química , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Formiatos/farmacología , Glucosa/farmacología , Glicerol/farmacología , Hidrólisis , Marcaje Isotópico , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/química , Fosforilación , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30 g g(-1)) and productivity (0.19 g L(-1) h(-1)). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3 g L(-1) resulted in high biomass production. The highest biomass concentration (21 g L(-1)), yield (0.45 g g(-1)) and productivity (0.31 g L(-1) h(-1)), as well as ribonucleotide production (13.13 mg g(-1)), were observed at 700 rpm and 0.5 vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.
Asunto(s)
Biocombustibles/análisis , Biomasa , Biotecnología/métodos , Glicerol/metabolismo , Ribonucleótidos/biosíntesis , Levaduras/metabolismo , Aerobiosis , Anaerobiosis , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos/microbiología , Ácidos Carboxílicos/metabolismo , Etanol/metabolismo , Redes y Vías Metabólicas , ARN/biosíntesisRESUMEN
The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B(12) ). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) enzyme that synthesizes the unique α-linked nucleotide N(1) -(5-phospho-α-D-ribosyl)-DMB (α-ribazole-5'-P, α-RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α-RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α-ribazole (α-R) transporter and an α-R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α-R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α-R in a Salmonella enterica cobT strain. LinCblT transported α-R across the cell membrane, but not α-RP or DMB. UV-visible spectroscopy and mass spectrometry data identified α-RP as the product of the ATP-dependent α-R kinase activity of LinCblS. Bioinformatics analyses suggest that α-R salvaging occurs in important Gram-positive human pathogens.
Asunto(s)
Listeria/enzimología , Complejos Multienzimáticos/metabolismo , Nucleotidiltransferasas/metabolismo , Pentosiltransferasa/metabolismo , Proteínas Quinasas/metabolismo , Ribonucleósidos/biosíntesis , Ribonucleótidos/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bencimidazoles , Clonación Molecular , Cobamidas/metabolismo , Biología Computacional , ADN Bacteriano/genética , Listeria/genética , Complejos Multienzimáticos/genética , Nucleotidiltransferasas/genética , Pentosiltransferasa/genética , Fosforilación , Plásmidos , Proteínas Quinasas/genética , Salmonella enterica/metabolismoRESUMEN
It has normally been assumed that ribonucleotides arose on the early Earth through a process in which ribose, the nucleobases, and phosphate became conjoined. However, under plausible prebiotic conditions, condensation of nucleobases with ribose to give beta-ribonucleosides is fraught with difficulties. The reaction with purine nucleobases is low-yielding and the reaction with the canonical pyrimidine nucleobases does not work at all. The reasons for these difficulties are considered and an alternative high-yielding synthesis of pyrimidine nucleotides is discussed. Fitting the new synthesis to a plausible geochemical scenario is a remaining challenge but the prospects appear good. Discovery of an improved method of purine synthesis, and an efficient means of stringing activated nucleotides together, will provide underpinning support to those theories that posit a central role for RNA in the origins of life.
Asunto(s)
Ribonucleótidos/química , Evolución Química , Origen de la Vida , Ribonucleótidos/biosíntesis , Ribonucleótidos/genéticaRESUMEN
Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non-small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.
Asunto(s)
Adenilato Quinasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Antagonistas del Ácido Fólico/uso terapéutico , Glutamatos/uso terapéutico , Guanina/análogos & derivados , Mesotelioma/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Quinasas/metabolismo , Ribonucleótidos/metabolismo , Ribonucleótidos/farmacología , Adenilato Quinasa/efectos de los fármacos , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , División Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glicina/análogos & derivados , Glicina/biosíntesis , Glicina/metabolismo , Guanina/uso terapéutico , Humanos , Cinética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Mesotelioma/patología , Pemetrexed , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Quinasas/efectos de los fármacos , Ribonucleótidos/biosíntesis , Serina-Treonina Quinasas TORRESUMEN
Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including 7 ATP-dependent enzymes, 2 amidotransferases and 2 tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and 2 to the PurM superfamily. The amidotransferases are unrelated, with one utilizing an N-terminal nucleophileglutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL.
Asunto(s)
Enzimas/química , Evolución Molecular , Modelos Moleculares , Purinas/biosíntesis , Sitios de Unión/genética , Enzimas/genética , Componentes del Gen , Inosina Monofosfato/biosíntesis , Estructura Molecular , Ribonucleótidos/biosíntesisRESUMEN
Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.
Asunto(s)
Proteínas Arqueales/metabolismo , Methanococcaceae/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Ribonucleótidos/biosíntesis , Proteínas Arqueales/química , Proteínas Arqueales/genética , Sitios de Unión , Cristalografía por Rayos X , Methanococcaceae/clasificación , Methanococcaceae/genética , Modelos Moleculares , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis.
Asunto(s)
Proteínas Bacterianas/genética , Lotus/metabolismo , Lotus/microbiología , Rhizobiaceae/genética , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Nódulos de las Raíces de las Plantas/microbiología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Mutación , Rhizobiaceae/metabolismo , Ribonucleótidos/biosíntesisRESUMEN
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. SmPurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The availability of a ternary complex enabled mapping of the active site, thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms.