RESUMEN
The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.
Asunto(s)
Antibacterianos , Inhibidores Enzimáticos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Relación Estructura-Actividad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Bacillus subtilis/efectos de los fármacos , Estructura Molecular , Relación Dosis-Respuesta a Droga , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Adenosina/síntesis química , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismo , HumanosRESUMEN
Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using a PS synthase 1 (PTDSS1) inhibitor and found that B cell lymphoma is highly dependent on PS. Inhibition of PTDSS1 in B cell lymphoma cells caused a reduction of PS and phosphatidylethanolamine levels and an increase of phosphoinositide levels. The resulting imbalance of the membrane phospholipidome lowered the activation threshold for B cell receptor (BCR), a B cell-specific survival mechanism. BCR hyperactivation led to aberrant elevation of downstream Ca2+ signaling and subsequent apoptotic cell death. In a mouse xenograft model, PTDSS1 inhibition efficiently suppressed tumor growth and prolonged survival. Our findings suggest that PS synthesis may be a critical vulnerability of malignant B cell lymphomas that can be targeted pharmacologically.
Asunto(s)
Linfoma de Células B , Fosfatidilserinas , Receptores de Antígenos de Linfocitos B , Animales , Humanos , Ratones , Apoptosis , Linfoma de Células B/genética , Fosfatidilserinas/biosíntesis , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Fosfatidilinositoles , Transferasas de Grupos Nitrogenados/antagonistas & inhibidoresRESUMEN
AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Acetilglucosamina/metabolismo , Cardiomegalia/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Transferasas de Grupos Nitrogenados/genética , Proteínas Quinasas Activadas por AMP/deficiencia , Acetilglucosamina/farmacología , Acilación/efectos de los fármacos , Animales , Animales Recién Nacidos , Azaserina/farmacología , Compuestos Azo/farmacología , Compuestos de Bifenilo , Cardiomegalia/metabolismo , Cardiomegalia/patología , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Regulación de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Glicosilación/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Masculino , Ratones , Ratones Noqueados , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismo , Norleucina/análogos & derivados , Norleucina/farmacología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Pironas/farmacología , Ratas , Ratas Wistar , Transducción de Señal , Tiofenos/farmacología , Troponina T/genética , Troponina T/metabolismoRESUMEN
Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/metabolismo , Unión ProteicaRESUMEN
In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) into Gln-tRNA(Gln) and Asn-tRNA(Asn), respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), with Ki values of 126 and 392µM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3'-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA(Gln) in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA(Gln) and/or Asn-tRNA(Asn) formation.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Dominio Catalítico , Cinética , Modelos Moleculares , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/aislamiento & purificación , Unión ProteicaRESUMEN
Plasmodium falciparum (PF) is the most severe malaria parasite. It is developing resistance quickly to existing drugs making it indispensable to discover new drugs. Effective drugs have been discovered targeting metabolic enzymes of the parasite. In order to predict new drug targets, computational methods can be used employing database information of metabolism. Using this data, we performed recently a computational network analysis of metabolism of PF. We analyzed the topology of the network to find reactions which are sensitive against perturbations, i.e., when a single enzyme is blocked by drugs. We now used a refined network comprising also the host enzymes which led to a refined set of the five targets glutamyl-tRNA (gln) amidotransferase, hydroxyethylthiazole kinase, deoxyribose-phophate aldolase, pseudouridylate synthase, and deoxyhypusine synthase. It was shown elsewhere that glutamyl-tRNA (gln) amidotransferase of other microorganisms can be inhibited by 6-diazo-5-oxonorleucine. Performing a half maximal inhibitory concentration (IC50) assay, we showed, that 6-diazo-5-oxonorleucine is also severely affecting viability of PF in blood plasma of the human host. We confirmed this by an in vivo study observing Plasmodium berghei infected mice.
Asunto(s)
Compuestos Azo/química , Malaria Falciparum/tratamiento farmacológico , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Norleucina/análogos & derivados , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Compuestos Azo/farmacología , Simulación por Computador , Eritrocitos/parasitología , Humanos , Ratones , Norleucina/química , Norleucina/farmacología , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , ARN de Transferencia de Alanina/síntesis química , ARN de Transferencia de Alanina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Transferasas de Grupos Nitrogenados/metabolismo , ARN de Transferencia de Alanina/química , Relación Estructura-ActividadRESUMEN
Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA(Gln) (Gln-tRNA(Gln)) and/or asparaginyl-tRNA(Asn) (Asn-tRNA(Asn)) are synthesized via an indirect pathway involving side chain amidation of misacylated glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) and/or aspartyl-tRNA(Asn) (Asp-tRNA(Asn)) by an amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active competitive inhibitor of the transamidase activity with respect to Asp-tRNA(Asn) (K(m)=2µM), with a K(i) value of 27µM.
Asunto(s)
Antibacterianos/química , Cloranfenicol/química , Inhibidores Enzimáticos/química , Helicobacter pylori/enzimología , Metionina/análogos & derivados , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Propanolaminas/química , Antibacterianos/síntesis química , Antibacterianos/farmacología , Cloranfenicol/síntesis química , Cloranfenicol/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Metionina/síntesis química , Metionina/química , Metionina/farmacología , Transferasas de Grupos Nitrogenados/metabolismo , Propanolaminas/síntesis química , Propanolaminas/farmacologíaRESUMEN
In eubacteria that lack glutaminyl-tRNA synthetase (GlnRS), a tRNA-dependent amidotransferase (GatCAB) recognizes mischarged Glu-tRNA(Gln) and converts it into Gln-tRNA(Gln). An inhibitor specific for GatCAB could therefore act as an antibiotic with a novel mode of action against multidrug-resistant bacteria such as Staphylococcus strains. However, there is no rapid, simple and efficient screening method for specifically monitoring the inhibition of GatCAB activity. We have focused on developing a simple system for monitoring the inhibition of GatCAB activity using Escherichia coli Top10 co-expressing the ndGluRS and GatCAB genes from Staphylococcus aureus Mu50. First, growth repression was confirmed by introducing ndgluRS from S. aureus Mu50 into E. coli. Then, we verified that co-expression of the gatCAB operon alleviated growth repression in the host E. coli. The screening system consisted of these two transformants and non-expressing E. coli Top10. The transformant harbors both ndGluRS gene and GatCAB operon could be co-expressed in the presence and in the absence of chemical compound of interest. Since there is no inhibitor that inactivates GatCAB activity, we expressed two inactive GatCAB deletion variants, GatCAB(Delta10) and GatCAB(DeltaCHD) together with ndGluRS in E. coli Top10. The expressed E. coli showed repressed growth as well as ndGluRS expressed. These results indicate that if GatCAB activity is inhibited in this co-expressed E. coli, the inhibition can be monitored by the decrease in O.D. of the co-expressed E. coli.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Nefelometría y Turbidimetría/métodos , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Staphylococcus aureus/enzimología , Clonación Molecular , Recuento de Colonia Microbiana , Escherichia coli/genética , Eliminación de Gen , Expresión Génica , Viabilidad Microbiana , Staphylococcus aureus/genéticaAsunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Puromicina/análogos & derivados , Puromicina/farmacología , Proteínas Bacterianas/química , Unión Competitiva/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Cinética , Estructura Molecular , Transferasas de Grupos Nitrogenados/química , Puromicina/química , EstereoisomerismoRESUMEN
The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.
Asunto(s)
Aminoaciltransferasas/metabolismo , Aspartato-ARNt Ligasa/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Aminoglicósidos/metabolismo , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidoresRESUMEN
Changes in glucose levels are known to directly alter gene expression. A number of previous studies have found that these effects are in part mediated by modulating the levels and the activity of transcription factors. We have investigated an alternative mechanism by which glucose might regulate gene expression by modulating levels of a transcriptional repressor. We have focused on Id2, which is a protein that indirectly regulates gene expression by sequestering certain transcription factors and preventing them from forming functional dimers. Id2 targets include the class A basic helix-loop-helix transcription factors and the sterol regulatory element-binding protein (SREBP)-1. We demonstrate that increases in glucose levels cause a rapid increase in levels of Id2 in J774.2 macrophages, and a number of lines of evidence indicate that this is via the hexosamine pathway because 1) the effect of glucose requires glutamine; 2) the effect of glucose is mimicked by low levels of glucosamine; 3) the effect of glucose is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT); and 4) adenoviral mediated overexpression of GFAT increases levels of Id2. We go on to show that increases in Id2 can have functional effects on metabolic genes, because Id2 blocked the SREBP-1-induced induction of hormone-sensitive lipase (HSL) promoter activity, whereas Id2 alone does not modulate activity of the HSL promoter. In summary, these studies define a new mechanism by which glucose uses the hexosamine pathway to regulate gene expression by increasing levels of a transcriptional repressor.
Asunto(s)
Glucosa/metabolismo , Glucosa/farmacología , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Macrófagos/metabolismo , Proteínas Represoras/metabolismo , Animales , Azaserina/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Glucosamina/metabolismo , Glutamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Hexosaminas/biosíntesis , Hexosaminas/metabolismo , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismo , Esterol Esterasa/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the action of PtdSer synthase (PSS) 1 and 2, which catalyze the conversion of phosphatidylcholine and phosphatidylethanolamine, respectively, to PtdSer. The PtdSer synthesis in intact cells and an isolated membrane fraction is inhibited by exogenous PtdSer, indicating that inhibition of PtdSer synthases by PtdSer is important for the regulation of PtdSer biosynthesis. In this study, to examine whether the inhibition occurs through the direct interaction of PtdSer with the synthases or is mediated by unidentified factor(s), we purified a FLAG and HA peptide-tagged form of Chinese hamster PSS 2 to near homogeneity. The purified enzyme, as well as the crude enzyme in a membrane fraction, was inhibited on the addition of PtdSer to the enzyme assay mixture. In contrast to PtdSer, phosphatidylcholine and phosphatidylethanolamine did not significantly inhibit the purified enzyme. Furthermore, PtdSer-resistant PtdSer synthesis was observed on cell-free assaying of the membrane fraction prepared from a Chinese hamster ovary cell strain whose PtdSer synthesis in vivo is not inhibited by exogenous PtdSer. These results suggested that the interaction of PtdSer with PSS 2 or a very minor protein co-purified with PSS 2 was critical for the regulation of PSS 2 activity in intact cells.
Asunto(s)
Cricetulus , Transferasas de Grupos Nitrogenados/aislamiento & purificación , Transferasas de Grupos Nitrogenados/metabolismo , Animales , Células CHO , Sistema Libre de Células , Cricetinae , Cinética , Sondas Moleculares , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Fosfatidilcolinas/farmacología , Fosfatidiletanolaminas/farmacología , Fosfatidilserinas/biosíntesis , Fosfatidilserinas/farmacología , Serina/farmacologíaRESUMEN
The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.
Asunto(s)
Antranilato Sintasa , Hexosaminas/biosíntesis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas/metabolismo , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Azaserina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glicosilación , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismoRESUMEN
Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.
Asunto(s)
Aspartato-ARNt Ligasa , ARN Bacteriano/biosíntesis , Aminoacil-ARN de Transferencia/biosíntesis , Thermus thermophilus/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Codón/genética , ADN Bacteriano/genética , Evolución Molecular , Datos de Secuencia Molecular , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Biosíntesis de Proteínas , Eliminación de Secuencia , Temperatura , Thermus thermophilus/genéticaRESUMEN
The absence of Gln-tRNA synthetase in certain bacteria necessitates an alternate pathway for the production of Gln-tRNA(Gln): misacylated Glu-tRNA(Gln) is transamidated by a Gln-dependent amidotransferase (Glu-AdT) via catalysis of Gln hydrolysis, ATP hydrolysis, activation of Glu-tRNA(Gln), and aminolysis of activated tRNA by Gln-derived NH(3). As observed for other Gln-coupled amidotransferases, substrate binding, Gln hydrolysis, and transamidation by Glu-AdT are tightly coordinated [Horiuchi, K. Y., Harpel, M. R., Shen, L., Luo, Y., Rogers, K. C., and Copeland, R. A. (2001) Biochemistry 40, 6450-6457]. However, Glu-AdT does not employ an active-site Cys nucleophile for Gln hydrolysis, as is common in all other glutaminases: some Glu-AdT lack Cys, but all contain a conserved Ser (Ser176 in the A subunit of Streptococcus pyogenes Glu-AdT) within a sequence signature motif of Ser-based amidases. Our current results with S. pyogenes Glu-AdT support this characterization of Glu-AdT as a Ser-based glutaminase. Slow-onset (approximately 50 M(-1) s(-1)), tight-binding (t(1/2) > 2.5 h for complex dissociation), Gln-competitive inhibition of the Glu-tRNA(Gln)/ATP-independent glutaminase activity of Glu-AdT by gamma-Glu boronic acid is consistent with engagement of a Ser nucleophile in the glutaminase active site. Conversion to rapidly reversible, yet still potent (K(i) = 73 nM) and Gln-competitive, inhibition under full transamidation conditions mirrors the coupling between Gln hydrolysis and aminolysis reactions during productive transamidation. Site-directed replacement of Ser176 by Ala abolishes glutaminase and Gln-dependent transamidase activities of Glu-AdT (>300-fold), but retains a wild-type level of NH(3)-dependent transamidation activity. These results demonstrate the essentiality of Ser176 for Gln hydrolysis, provide additional support for coordinated coupling of Gln hydrolysis and transamidase transition states during catalysis, and validate glutaminase-directed inhibition of Glu-AdT as a route for antimicrobial chemotherapy.
Asunto(s)
Glutaminasa/química , Mutagénesis Sitio-Dirigida , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/genética , Serina/química , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Alanina/genética , Amidas/antagonistas & inhibidores , Amidas/metabolismo , Secuencia de Aminoácidos , Aminoaciltransferasas/química , Sitios de Unión/genética , Unión Competitiva/genética , Ácidos Borónicos/antagonistas & inhibidores , Ácidos Borónicos/química , Secuencia Conservada , Glutamina/antagonistas & inhibidores , Glutamina/metabolismo , Hidrólisis/efectos de los fármacos , Datos de Secuencia Molecular , Transferasas de Grupos Nitrogenados/metabolismo , Serina/genéticaRESUMEN
Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.
Asunto(s)
Antranilato Sintasa , Antibacterianos/farmacología , Carbamoil-Fosfato Sintasa (Amoniaco)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Isoxazoles/farmacología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Carbamoil-Fosfato Sintasa (Amoniaco)/química , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Cristalografía por Rayos X , Escherichia coli/enzimología , Cinética , Modelos Moleculares , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/metabolismo , Conformación ProteicaRESUMEN
Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.
Asunto(s)
Antranilato Sintasa , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Isoxazoles/farmacología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/genética , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva , Medios de Cultivo , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Perfilación de la Expresión Génica/métodos , Glutamina/metabolismo , Imidazoles/metabolismo , Isoxazoles/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Ribonucleótidos/metabolismo , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.