RESUMEN
Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. Herein, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of how primary and secondary metabolism are interlinked.
Asunto(s)
Formicinas/biosíntesis , Purinas/biosíntesis , Pirazoles/metabolismo , Pirimidinas/biosíntesis , Streptomyces/química , Formicinas/química , Formicinas/metabolismo , Conformación Molecular , Familia de Multigenes , Purinas/química , Pirazoles/química , Pirimidinas/química , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
A steady-state absorption and emission spectroscopy was used to create a comprehensive work and to study the interaction of the wild type Escherichia coli purine nucleoside phosphorylase and its mutants, PNPF159Y and PNPF159A, with a potent E. coli PNP inhibitor - formycin A. The absorption and emission spectra were recorded in the presence and absence of the phosphate at the 50â¯mM concentration. From the collected sets of data dissociation constants (Kd), apparent dissociation constants (Kapp) and Hill's coefficients (h) were calculated. Additionally, the temperature dependence of the enzymes emission quenching at two temperatures, 10⯰C and 25⯰C, was examined. To verify the calculations, total difference absorption spectra were computed for all types of the complexes. A prominent quenching of the PNPF159Y emission indicates a complex formation, with the strongest association in the phosphate buffer, pH 7, relative to the wild type enzyme. On the other hand, results testify to a deterioration of the interactions in the E. coli PNP/PNPF159Y and formycin A complexes in the presence of the phosphate, pH 8.3. Moreover, data obtained for the PNPF159A-FA complexes confirm a weak association of the FA to the mutant's active center.
Asunto(s)
Sustitución de Aminoácidos , Escherichia coli/enzimología , Formicinas/metabolismo , Fenilalanina , Fosfatos/farmacología , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Concentración de Iones de Hidrógeno , Mutación , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Purina-Nucleósido Fosforilasa/químicaRESUMEN
The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λex 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Formicinas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Formicinas/química , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/genética , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the "standard" phosphate binding site.
Asunto(s)
Antineoplásicos/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Formicinas/metabolismo , Fosfatos/metabolismo , Purina-Nucleósido Fosforilasa/química , Antineoplásicos/química , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Formicinas/química , Cinética , Ligandos , Modelos Moleculares , Concentración Osmolar , Fosfatos/química , Conformación Proteica , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Volumetría , Agua/química , Agua/metabolismoRESUMEN
We previously reported that both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters are expressed in the human liver (21). Here we report a study that investigated the expression of these transporters (transcripts and proteins) and their role in the hepatobiliary transport of nucleosides/nucleoside drugs using sandwich-cultured human hepatocytes. In the hepatic tissue, the rank order of the mRNA expression of the transporters was hCNT1 approximately hENT1>hENT2 approximately hCNT2>hCNT3. In sandwich-cultured hepatocytes, the mRNA expression of hCNT2 and hENT2 was comparable to that in hepatic tissue, whereas the expression of corresponding transporters in the two-dimensional hepatocyte cultures was lower. Colocalization studies demonstrated predominant localization of these transporters at the sinusoidal membrane and of hENT1, hCNT1, and hCNT2 at the canalicular membrane. In the sandwich-cultured hepatocytes, ENTs were the major contributors to the transport of thymidine (hENT1, 63%; hENT2, 23%) or guanosine (hENT1, 53%; hENT2, 24%) into the hepatocytes followed by hCNT1 (10%) for thymidine or hCNT2 (23%) for guanosine. Although ribavirin was predominately transported (89%) into the hepatocytes by hENT1, fialuridine (FIAU) was transported by both hENT1 (30%) and hCNTs (61%). The extensively metabolized natural nucleosides were not effluxed into the bile, whereas significant biliary-efflux was observed of FIAU (19%), ribavirin (30%), and formycin B (35%). We conclude that the hepatic activity of hENT1 and hCNT1/2 transporters will determine the in vivo hepatic distribution and therefore the efficacy and/or toxicity of nucleoside drugs used to treat hepatic diseases.
Asunto(s)
Bilis/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Hepatocitos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Nucleósidos/metabolismo , Adulto , Anciano , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/metabolismo , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Células Cultivadas , Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Femenino , Formicinas/metabolismo , Guanosina/metabolismo , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , ARN Mensajero/metabolismo , Ribavirina/metabolismo , Timidina/metabolismoRESUMEN
5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) and plays a key role in four metabolic processes: biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. The absence of the nucleosidase in mammalian species has implicated this enzyme as a target for antimicrobial drug design. MTAN from the pathogenic bacterium Staphylococcus aureus (SaMTAN) has been kinetically characterized and its structure has been determined in complex with the transition-state analogue formycin A (FMA) at 1.7 A resolution. A comparison of the SaMTAN-FMA complex with available Escherichia coli MTAN structures shows strong conservation of the overall structure and in particular of the active site. The presence of an extra water molecule, which forms a hydrogen bond to the O4' atom of formycin A in the active site of SaMTAN, produces electron withdrawal from the ribosyl group and may explain the lower catalytic efficiency that SaMTAN exhibits when metabolizing MTA and SAH relative to the E. coli enzyme. The implications of this structure for broad-based antibiotic design are discussed.
Asunto(s)
Desoxiadenosinas/química , N-Glicosil Hidrolasas/química , S-Adenosilhomocisteína/metabolismo , Staphylococcus aureus/enzimología , Tionucleósidos/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalización , Desoxiadenosinas/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Formicinas/metabolismo , Enlace de Hidrógeno , Cinética , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismo , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Tionucleósidos/metabolismoRESUMEN
Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with Ki = 100 micro M and more effectively by its aglycone (7KPP), IC50 35-100 micro M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with Ki approximately 2-4 micro M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.
Asunto(s)
Formicinas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Bazo/enzimología , Animales , Bovinos , Formicinas/química , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Cinética , Pirimidinas/química , Pirimidinas/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.
Asunto(s)
Quinasa de la Caseína II/fisiología , Tranportador Equilibrativo 1 de Nucleósido/fisiología , Transportador Equilibrativo 2 de Nucleósido/fisiología , Tioinosina/análogos & derivados , 2-Cloroadenosina/metabolismo , Neoplasias Óseas/enzimología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Quinasa de la Caseína II/deficiencia , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Línea Celular Tumoral , Sistemas de Computación , Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Formicinas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Nucleósidos/metabolismo , Osteosarcoma/enzimología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Reacción en Cadena de la Polimerasa/métodos , Especificidad por Sustrato , Tioinosina/metabolismo , Transfección/métodos , Tritio/metabolismoRESUMEN
1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.
Asunto(s)
Células Endoteliales/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Transporte de Nucleósidos/biosíntesis , Proteínas de Transporte de Nucleósidos/fisiología , Tioinosina/análogos & derivados , Animales , Capilares/citología , Capilares/metabolismo , Separación Celular , Células Cultivadas , Cartilla de ADN , Dilazep/farmacología , Dipiridamol/farmacología , Formicinas/metabolismo , Músculo Esquelético/citología , Piperazinas/farmacología , Ensayo de Unión Radioligante , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tioinosina/metabolismo , Vasodilatadores/farmacologíaRESUMEN
We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.
Asunto(s)
Proteínas Portadoras/genética , Tranportador Equilibrativo 1 de Nucleósido/genética , Tioinosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Dilazep/farmacología , Dipiridamol/farmacología , Perros , Relación Dosis-Respuesta a Droga , Tranportador Equilibrativo 1 de Nucleósido/química , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Formicinas/metabolismo , Cinética , Datos de Secuencia Molecular , Piperazinas/farmacología , Unión Proteica/efectos de los fármacos , Conformación Proteica , Ensayo de Unión Radioligante , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Tioinosina/metabolismo , TritioRESUMEN
Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale-up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different environments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root-packing density. Surprisingly, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production.
Asunto(s)
Reactores Biológicos , Modelos Biológicos , Raíces de Plantas/metabolismo , Terpenos/síntesis química , Isomerasas Aldosa-Cetosa/metabolismo , Transferasas Alquil y Aril/metabolismo , Artemisia annua/enzimología , Artemisia annua/genética , Artemisia annua/metabolismo , Artemisininas/síntesis química , Técnicas de Cultivo de Célula , Medios de Cultivo Condicionados , Formicinas/metabolismo , Expresión Génica , Geraniltranstransferasa , Hemiterpenos/biosíntesis , Hemiterpenos/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Luz , Complejos Multienzimáticos/metabolismo , Compuestos Organofosforados/metabolismo , Oxidorreductasas/metabolismo , Oxígeno , Raíces de Plantas/enzimología , Ribonucleótidos/metabolismo , Sesquiterpenos/síntesis química , Terpenos/metabolismo , Transferasas/metabolismoRESUMEN
A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl-L-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.
Asunto(s)
Fluorescencia , Formicinas/química , Modelos Químicos , Espectrometría de Fluorescencia/métodos , Triptófano/análogos & derivados , Triptófano/química , Tirosina/química , Simulación por Computador , Formicinas/análisis , Formicinas/metabolismo , Semivida , Luz , Triptófano/análisis , Triptófano/efectos de la radiación , Tirosina/análisis , Tirosina/efectos de la radiaciónRESUMEN
The mechanism responsible for the reduced clearance of benzylpenicillin (BPC) from the cerebrospinal fluid (CSF) was investigated in rats that received an intracisternal administration of lipopolysaccharide (LPS). BPC was intraventricularly injected and its elimination from the CSF studied. During the inflammation created by the LPS administration to the cisterna magna, the clearance of BPC and taurine from the CSF was significantly reduced but reverted to the control level when N-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, was intracisternally administered. The in vitro uptake of BPC and taurine was significantly reduced in the choroid plexus (CP, the blood-CSF barrier) of rats with experimental inflammation and in control CP that had been pretreated with sodium nitroprusside (SNP, an NO donor). Interestingly, the clearance and CP uptake of formycin B, a substrate for a nucleoside transporter, were not affected by the experimental inflammation or by pretreatement with SNP. These observations suggest that the BPC transporter, and probably other transport systems as well, is functionally sensitive to NO in the blood-CSF barrier. Therefore, functional impairment of BPC transport in the CP by NO may be partly responsible for the increase in BPC concentration in the CSF during inflammation such as that caused by meningitis.
Asunto(s)
Lipopolisacáridos/farmacología , Penicilina G/líquido cefalorraquídeo , Penicilinas/líquido cefalorraquídeo , Algoritmos , Animales , Encéfalo/metabolismo , Líquido Cefalorraquídeo/citología , Plexo Coroideo/metabolismo , Cisterna Magna , Formicinas/metabolismo , Recuento de Leucocitos , Lipopolisacáridos/administración & dosificación , Masculino , Microinyecciones , Óxido Nítrico/líquido cefalorraquídeo , Óxidos de Nitrógeno/líquido cefalorraquídeo , Ratas , Ratas Sprague-Dawley , Taurina/metabolismoRESUMEN
The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.
Asunto(s)
Escherichia coli/enzimología , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Dimerización , Fluorescencia , Formicinas/química , Formicinas/metabolismo , Guanosina/química , Guanosina/metabolismo , Enlace de Hidrógeno , Inosina/química , Inosina/metabolismo , Cinética , Modelos Moleculares , Nucleósidos/química , Nucleósidos/metabolismo , Fosfatos/metabolismo , Estructura Cuaternaria de Proteína , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Relación Estructura-Actividad , Sulfatos/metabolismo , VolumetríaRESUMEN
Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.
Asunto(s)
Adenosina Trifosfato/metabolismo , Transportador Equilibrativo 2 de Nucleósido , Glioma/metabolismo , Nucleósidos/metabolismo , Purinas/metabolismo , Purinas/farmacocinética , Tioinosina/análogos & derivados , AMP Desaminasa/antagonistas & inhibidores , Adenina/metabolismo , Adenosina/metabolismo , Adenosina/farmacocinética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteínas de Transporte de Nucleósido Equilibrativas , Formicinas/metabolismo , Formicinas/farmacocinética , Glioma/patología , Hipoxantina/metabolismo , Inosina/metabolismo , Yodoacetatos/farmacología , Nucleósidos/farmacocinética , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismo , Cianuro de Sodio/farmacología , Células Tumorales CultivadasRESUMEN
The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.
Asunto(s)
Liasas de Carbono-Oxígeno/genética , ADN Glicosilasas , Reparación del ADN/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Glicósido Hidrolasas/genética , N-Glicosil Hidrolasas/genética , Mutación Puntual , Secuencia de Aminoácidos , Borohidruros/farmacología , Liasas de Carbono-Oxígeno/clasificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxiadenosinas/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/enzimología , Formicinas/metabolismo , Glicósido Hidrolasas/clasificación , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , N-Glicosil Hidrolasas/clasificación , Ingeniería de Proteínas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.
Asunto(s)
Formicinas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ribonucleótidos/metabolismo , Cristalografía por Rayos X , Formicinas/química , Hidrólisis , Estructura Molecular , Proteínas Proto-Oncogénicas p21(ras)/química , Ribonucleótidos/química , Temperatura , AguaRESUMEN
The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.
Asunto(s)
ADN Glicosilasas , ADN/metabolismo , Escherichia coli/enzimología , N-Glicosil Hidrolasas/metabolismo , Ácido Apurínico/genética , Ácido Apurínico/metabolismo , Disparidad de Par Base/genética , Secuencia de Bases , Sitios de Unión , ADN/genética , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Desoxiadenosinas/metabolismo , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Escherichia coli/genética , Formicinas/metabolismo , Especificidad por Sustrato , Ésteres del Ácido Sulfúrico/metabolismo , TermodinámicaRESUMEN
Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma membrane. In the present study, we examined the role of these transporters in the accumulation of gemcitabine by a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitibine was compared with that of [3H]uridine and [3H]formycin B in the parent cell line (HN-5a) and in a gemcitabine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equilibrative, inhibitor-sensitive) transporter subtype; less than 10% of the influx of [3H]formycin B or [3H]uridine was mediated by the ei (equilibrative inhibitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]Gemcitabine (10 microM) entered these cells via both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]formycin B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitabine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitabine. These results show that gemcitabine is a substrate for both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitabine resistance of the GEM-8e cells cannot be attributed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted because this system has the potential to significantly affect the clinical efficacy of gemcitabine.
Asunto(s)
Antimetabolitos Antineoplásicos/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas Portadoras/metabolismo , Desoxicitidina/análogos & derivados , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/metabolismo , Desoxicitidina/metabolismo , Resistencia a Medicamentos , Formicinas/metabolismo , Humanos , Proteínas de Transporte de Nucleósidos , Factores de Tiempo , Tritio , Células Tumorales Cultivadas , Uridina/metabolismo , GemcitabinaRESUMEN
The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2',2'-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 approximately 400 microM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.