RESUMEN
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Asunto(s)
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidad por Sustrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/químicaRESUMEN
The development of facile, low-cost reliable, and precise onsite assays for the bioactive component hypoxanthine (Hx) in meat products is significant for safeguarding food safety and public health. Herein, we proposed a smartphone-assissted aggregation-induced emission (AIE) fluorogen tetraphenylethene (TPE)-incorporated amorphous Fe-doped phosphotungstates (Fe-Phos@TPE) nanozyme-based ratiometric fluorescence-colorimetric dual-mode biosensor for achieving the onsite visual detection of Hx. When the Hx existed, xanthine oxidase (XOD) catalyzed Hx into H2O2 to be further catalyzed into â¢OH by the prominent peroxidase activity of Fe-Phos@TPE at pH = 6.5, resulting in the oxidization of nonfluorescent o-phenylenediamine (OPD, naked-eye colorless) to be yellow fluorescent emissive 2,3-diaminophenazine (DAP, naked-eye dark yellow) at 550 nm as well as the intrinsic blue fluorescence of Fe-Phos@TPE at 440 nm to be decreased via inner-filter effect (IFE) action, thereby realizing a multi-enzyme cascade catalytic reaction at near-neutral pH to overcome the traditional acidity dependence-induced time-consuming and low sensitivity troublesome.
Asunto(s)
Técnicas Biosensibles , Hipoxantina , Productos de la Carne , Técnicas Biosensibles/instrumentación , Hipoxantina/análisis , Hipoxantina/química , Productos de la Carne/análisis , Xantina Oxidasa/química , Xantina Oxidasa/metabolismo , Contaminación de Alimentos/análisis , Animales , Colorantes Fluorescentes/química , Fluorescencia , Teléfono Inteligente , Colorimetría/métodosRESUMEN
Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.
Asunto(s)
Técnicas Biosensibles , Colodión , Hipoxantina , Oligosacáridos , Xantina Oxidasa , Animales , Oligosacáridos/química , Oligosacáridos/análisis , Técnicas Biosensibles/métodos , Hipoxantina/análisis , Hipoxantina/química , Colodión/química , Xantina Oxidasa/química , Xantina Oxidasa/metabolismo , Peces , Quitina/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Tecnología Química Verde/métodos , Propiedades de Superficie , Límite de DetecciónRESUMEN
The research in the field of biosensors has recently been focused on the design and development of functional electrode materials that can respond to changes in their biochemical environment. Here, we report the synthesis of dicalcium phosphate dihydrate (DCPD), also known as brushite (CaHPO4·2H2O) by soft chemical method and its application for electrochemical sensing of four different analytes. Phase purity, structure, chemical composition and surface morphology of the synthesized nanoparticles have been investigated using powder XRD, FTIR, SEM, XPS and HRTEM methods. Electrochemical sensor was prepared by modifying GCE with brushite and the modified electrodes were successfully used for either independent or simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine in their mixture. The brushite/GCE exhibited four strong well-defined separate peaks corresponding to the oxidation of UA, XN, HXN and CF in phosphate buffer saline (PBS) at pH 7.4. The fabricated electrode showed low detection limits (S/N = 3) of 0.576, 1.0, 0.076 and 1.26 µM for UA, XN, HXN and CF respectively. Practical application of the fabricated electrode has been demonstrated by determining UA, XN, HXN and CF in human urine and coffee samples by direct method. The brushite offers scope for fabrication of sensor systems for implantable medical applications.
Asunto(s)
Nanopartículas , Ácido Úrico , Humanos , Xantina/química , Xantina/orina , Hipoxantina/química , Hipoxantina/orina , Ácido Úrico/orina , Cafeína , Electrodos , Técnicas Electroquímicas , Ácido AscórbicoRESUMEN
Photoelectrochemical (PEC) enzymatic biosensors integrate the excellent selectivity of enzymes and high sensitivity of PEC bioanalysis, but the drawbacks such as high cost, poor stability, and tedious immobilization of natural enzymes on photoelectrodes severely suppress their applications. AgCu@CuO aerogel-based photoelectrode materials with both remarkable enzyme-like activities and outstanding photoelectric properties were innovatively designed and synthesized to evaluate the activity of xanthine oxidase with a wide linear detection range and a low limit of detection.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Geles/química , Xantina Oxidasa/análisis , Técnicas Biosensibles/instrumentación , Catálisis , Cobre/química , Técnicas Electroquímicas/instrumentación , Electrodos , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Peróxido de Hidrógeno/química , Hipoxantina/química , Límite de Detección , Naftoles/química , Oxidación-Reducción , Procesos Fotoquímicos , Plata/química , Xantina Oxidasa/químicaRESUMEN
Cyclic dinucleotides (CDNs) could activate stimulator of interferon genes (STING) protein to produce type I interferon and other pro-inflammation cytokines in mammalian cells. To explore new types of potentially efficient STING activators targeting all five major hSTING variants (WT, R232H, HAQ, AQ and R293Q), we here reported the synthesis of a total of 19 inosine-containing CDNs based on the combinations of hypoxanthine with four natural bases (A, G, C and U) and three phosphodiester linkage backbones (3'-3', 2'-3', 2'-2'). The IFN-ß induction results showed that all of the 2'-3' and 2'-2' CDNs linked by inosine and purine nucleosides favored the stacking interaction with Y167 and R238 residues of hSTING protein, and several CDNs constructed by hypoxanthine and pyrimidine like c[I(2',5')U(2',5')] could also activate all five hSTING variants. The molecular dynamic simulation and the isothermal titration calorimetric (ITC) assay further demonstrated the potential of cAIMP isomers with 2'-5' phosphate to form the hydrogen binding with R232 and R238 residues of hSTING in an entropically driven manner compared to cGAMP isomers. It would be promising to exploit novel inosine-mixed CDNs as activators of hSTING variants in immune therapy.
Asunto(s)
GMP Cíclico/química , GMP Cíclico/metabolismo , Fosfatos de Dinucleósidos/química , Inosina/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Citocinas/metabolismo , Diseño de Fármacos , Humanos , Hipoxantina/química , Isomerismo , Simulación del Acoplamiento Molecular , Unión Proteica , Pirimidinas/química , Transducción de Señal , Relación Estructura-ActividadRESUMEN
This work reports a new method to evaluate the antioxidant capacity of infusions and beverages, based on superoxide radicals. Radicals produced by the enzymatic reaction between acetylcholinesterase and hypoxanthine oxidized antioxidant molecules present in commercially available samples or standard solutions, which was monitored by means of cyclic voltammetry using a carbon paste electrode. The Trolox equivalent antioxidant capacity (TEAC) of red wine, coffee and green tea determined using this method were: (1.20 ± 0.06), (0.90 ± 0.02), and (0.65 ± 0.02), respectively. This method suggested TEACred wine > TEACcoffee > TEACgreen tea, which is the same as DPPH, spectrophotometric method. However, the electrochemical one proposed here is rapid and simple.
Asunto(s)
Antioxidantes/química , Bebidas/análisis , Técnicas Electroquímicas/métodos , Superóxidos/química , Antioxidantes/metabolismo , Café/química , Electrodos , Concentración de Iones de Hidrógeno , Hipoxantina/química , Hipoxantina/metabolismo , Oxidación-Reducción , Superóxidos/metabolismo , Té/química , Vino/análisis , Xantina Oxidasa/metabolismoRESUMEN
We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar structures. The investigated compounds were either free forms or derivatives of adenine, guanine, hypoxanthine, thymine, uracil and cytosine. We divided our findings into categories including types of pairs, protonation patterns and whether they are formed by free bases or substituted ones. We found base pair types that are exclusive to small molecule crystal structures, some that can be found only in RNA containing crystal structures and many that are native to both environments. With a few exceptions, nucleobase protonation generally followed a standard pattern governed by pKa values. The lengths of hydrogen bonds did not depend on whether the nucleobases forming a base pair were charged or not. The reasons why particular nucleobases formed base pairs in a certain way varied significantly.
Asunto(s)
Bases de Datos de Proteínas , Enlace de Hidrógeno , Conformación Proteica , Proteínas/genética , Adenina/química , Emparejamiento Base/genética , Cristalografía por Rayos X , Citosina/química , Guanina/química , Hipoxantina/química , Estructura Molecular , Proteínas/química , Proteínas/ultraestructura , Bibliotecas de Moléculas Pequeñas/química , Timina/química , Uracilo/químicaRESUMEN
The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide.
Asunto(s)
Acetamidas/farmacología , Antibacterianos/farmacología , Hipoxantina/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Ribosómicas/antagonistas & inhibidores , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Acetamidas/síntesis química , Acetamidas/química , Antibacterianos/síntesis química , Antibacterianos/química , Descubrimiento de Drogas , Hipoxantina/síntesis química , Hipoxantina/química , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas Ribosómicas/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/metabolismoRESUMEN
The cyclo-l-Trp-l-Trp (cWW, 1) tailoring P450 GutD2774 from Streptomyces lavendulae was characterized by expression in Streptomyces coelicolor, precursor feeding and enzyme assays. GutD2774 catalyzes mainly the transfer of hypoxanthine to C2 and C3 of the indole ring of 1. cWW adducts with guanine were detected as minor products. An orthologous cluster was identified in Streptomyces xanthophaeus. These results expand the spectrum of cyclodipeptide derivatives by involvement of an additional nucleobase and identification of new coupling patterns.
Asunto(s)
Dipéptidos/química , Hipoxantina/química , Péptidos Cíclicos/química , Transferasas/química , Catálisis , Sistema Enzimático del Citocromo P-450/química , Estructura Molecular , Streptomyces/genética , Streptomyces/metabolismo , Transferasas/genética , Transferasas/metabolismoRESUMEN
The environmental stability, water-processibility and life-span of black phosphorene (BP) severely limit the application of its electronic devices in aqueous system containing oxygen. We reported the controllable preparation of in-situ reduction and deposition of silver nanoparticles on the BP surface and its amino-functionalized multi-walled carbon nanotubes (NH2-MWCNT) nanocomposite. With the addition of both NH2-MWCNT and Ag+, the BP-based nanocomposite was prepared by ultrasonic-assisted liquid-phase exfoliation and was dispersed in carboxymethyl cellulose sodium (CMC) aqueous solution. The morphology, microstructure, and electrochemical properties of the nanohybrid were characterized. NH2-MWCNT-BP-AgNPs showed high environmental stability, good water-processibility, satisfactory life-spans, superior electrocatalytic capacity with enzyme-like kinetic characteristics. The nanohybrid was applied as electrochemical sensors for single/simultaneous analysis of uric acid (UA), xanthine (XT) and hypoxanthine (HX). Excellent voltammetric responses for simultaneous determination in linear ranges of 0.1-800⯵M with a limit of detection (LOD) of 0.052⯵M for UA, 0.5-680⯵M with a LOD of 0.021⯵M for XT, and 0.7-320⯵M with a LOD of 0.025⯵M for HX under optimal conditions. Besides, the developed nanozyme sensor was employed for simultaneous voltammetric analysis of UA, XT and HX in real samples with acceptable recoveries. This work will provide theoretical guidance and experimental support for the preparation and application of two-dimensional materials, nanozymes and sensing devices.
Asunto(s)
Técnicas Biosensibles , Hipoxantina/aislamiento & purificación , Ácido Úrico/aislamiento & purificación , Agua/química , Xantina/aislamiento & purificación , Carboximetilcelulosa de Sodio/química , Técnicas Electroquímicas , Humanos , Hipoxantina/química , Límite de Detección , Nanopartículas del Metal/química , Nanocompuestos/química , Nanotubos de Carbono/química , Oxígeno/química , Plata/química , Ácido Úrico/química , Xantina/químicaRESUMEN
The synthesis of nucleobases in natural environments, especially in interstellar molecular clouds, is the focus of a long-standing debate regarding prebiotic chemical evolution. Here we report the simultaneous detection of all three pyrimidine (cytosine, uracil and thymine) and three purine nucleobases (adenine, xanthine and hypoxanthine) in interstellar ice analogues composed of simple molecules including H2O, CO, NH3 and CH3OH after exposure to ultraviolet photons followed by thermal processes, that is, in conditions that simulate the chemical processes accompanying star formation from molecular clouds. Photolysis of primitive gas molecules at 10 K might be one of the key steps in the production of nucleobases. The present results strongly suggest that the evolution from molecular clouds to stars and planets provides a suitable environment for nucleobase synthesis in space.
Asunto(s)
Adenina/química , Citosina/química , Hipoxantina/química , Timina/química , Uracilo/química , Xantina/química , Adenina/síntesis química , Amoníaco/química , Monóxido de Carbono/química , Citosina/síntesis química , Evolución Química , Medio Ambiente Extraterrestre , Hipoxantina/síntesis química , Hielo , Metanol/química , Estructura Molecular , Procesos Fotoquímicos/efectos de la radiación , Timina/síntesis química , Rayos Ultravioleta , Uracilo/síntesis química , Agua/química , Xantina/síntesis químicaRESUMEN
Hypoxanthine riboside (HXR) is a nucleoside essential for wobble base pairs to translate the genetic code. In this work, an absorption and luminescence study showed that HXR and human serum albumin (HSA) formed a new complex through hydrogen bonds and van der Waals forces at ground state. Fluorescence probe experiments indicated that HXR entered the first subdomain of domain II in HSA and was fixed by amino acid residues in site I defined by Sudlow, and after competing with a known site marker. The recognition interaction featured negative ΔHÏ´ , ΔSÏ´ and ΔGÏ´ thermodynamic parameters. Fluorescence and circular dichroism spectra described the polarity of residues and α-helix and ß-strand content changed because of HXR binding. The most rational structure for the HXR-HSA complex was recommended by the molecular docking method, in which the binding location, molecular orientation, adjacent amino acid residues, and hydrogen bonds were included. In addition, the influence of ß-cyclodextrin and some essential metal ions on the balance of the HSA-HXR system interaction was measured. The study gained comprehensive information on the transportation mechanism for HXR in blood, and was of great significance in understanding the theory of HXR biotransformation and in discussing its clinical in vivo half-life.
Asunto(s)
Hipoxantina/química , Simulación del Acoplamiento Molecular , Humanos , Albúmina Sérica Humana/química , Espectrometría de FluorescenciaRESUMEN
The paper describes the development of an amperometric biosensor using Prussian Blue (PB) modified electrodes containing xanthine oxidase (XOD). The enzyme is immobilized by photo-polymerization into an azide-unit pendant water-soluble photopolymer (PVA-AWP). The parameters of the fabrication of the biosensor, XOD:PVA/AWP ratio, crosslinking irradiation time, and XOD charge, were optimized. Operational conditions for electrode preparation were defined as 1:2 ratio of XOD:PVA/AWP; exposure time to neon light of 30 â¯min; pHâ¯=â¯7.5⯠at room temperature and enzymatic charge of 8 mU per electrode. The biosensors showed stable, fast, simple, selective, cost-effective and sensitive (-2.72E-8 â¯Aâ¯mol L-1), with a good linear range (1.0-75⯠µmolâ¯L-1), and respectively detection and quantification limits for antioxidants of 2.17, and 7.15⯠µmolâ¯L-1. The applicability of this biosensor was demonstrated by in vitro analysis of gallic acid as standard antioxidant and Amazonian fruits as natural sources.
Asunto(s)
Antioxidantes/análisis , Técnicas Biosensibles/métodos , Frutas/química , Xantina Oxidasa/química , Antioxidantes/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Enzimas Inmovilizadas/química , Ferrocianuros/química , Peróxido de Hidrógeno/química , Hipoxantina/química , Límite de Detección , Oxidación-ReducciónRESUMEN
Nucleotides that contain two nucleobases (double-headed nucleotides) have the potential to condense the information of two separate nucleotides into one. This presupposes that both bases must successfully pair with a cognate strand. Here, double-headed nucleotides that feature cytosine, guanine, thymine, adenine, hypoxanthine, and diaminopurine linked to the C2'-position of an arabinose scaffold were developed and examined in full detail. These monomeric units were efficiently prepared by convergent synthesis and incorporated into DNA oligonucleotides by means of the automated phosphoramidite method. Their pairing efficiency was assessed by UV-based melting-temperature analysis in several contexts and extensive molecular dynamics studies. Altogether, the results show that these double-headed nucleotides have a well-defined structure and invariably behave as functional dinucleotide mimics in DNA duplexes.
Asunto(s)
Emparejamiento Base , Nucleótidos/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , Adenina/química , Disparidad de Par Base , Citosina/química , ADN/química , Guanina/química , Hipoxantina/química , Modelos Moleculares , Conformación de Ácido Nucleico , Timina/químicaRESUMEN
Our laboratories have been investigating synthetic analogues of marine alkaloid rigidins that possess promising anticancer activities. These analogues, based on the 7-deazahypoxanthine skeleton, are available in one- or two-step synthetic sequences and exert cytotoxicity by disrupting microtubule dynamics in cancer cells. In the present work we extended the available structure-activity relationship (SAR) data to N3- and N9-substituted derivatives. Although N3 substitution results in loss of activity, the N9-substituted compounds retain nanomolar antiproliferative activities and the anti-tubulin mode of action of the original unsubstituted compounds. Furthermore, our results also demonstrate that multidrug-resistance (MDR) proteins do not confer resistance to both N9-unsubstituted and -substituted compounds. It was found that sublines overexpressing ABCG2, ABCC1, and ABCB1 proteins are as responsive to the rigidin analogues as their parental cell lines. Thus, the study reported herein provides further impetus to investigate the rigidin-inspired 7-deazahypoxanthines as promising anticancer agents.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Alcaloides/farmacología , Antineoplásicos/farmacología , Hipoxantina/farmacología , Microtúbulos/efectos de los fármacos , Pirimidinas/farmacología , Pirroles/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Alcaloides/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Hipoxantina/síntesis química , Hipoxantina/química , Células MCF-7 , Células de Riñón Canino Madin Darby/efectos de los fármacos , Microtúbulos/metabolismo , Estructura Molecular , Pirimidinas/química , Pirroles/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
In the present work, the influence of Cu+ binding to N3- and N7-positions of hypoxanthine on energetic, geometrical and topological properties of hypoxanthine-guanine, hypoxanthine-adenine, hypoxanthine-cytosine, hypoxanthine-thymine and hypoxanthine-hypoxanthine mismatches is theoretically investigated. The calculations, in gas phase, are performed at B3LYP/6-311++G(3df,3pd) level of theory. Unlike the other mispairs, Cu+ binding to N3-position of hypoxanthine causes the proton transfer process from enol form of hypoxanthine to imino forms of adenine and cytosine. This process also occurs in all mismatches having enol form of hypoxanthine when Cu+ binds to N7-position of hypoxanthine. The mismatches are stabilized by hydrogen bonds. The influence of Cu+ on hydrogen bonds is also examined by atoms in molecules (AIM) and natural bond orbital (NBO) analyses. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Disparidad de Par Base , Cobre/metabolismo , ADN/metabolismo , Teoría Funcional de la Densidad , Hipoxantina/metabolismo , Cobre/química , ADN/química , Enlace de Hidrógeno , Hipoxantina/química , Iones , Modelos Moleculares , Conformación Molecular , Protones , TermodinámicaRESUMEN
Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine-uridine nucleoside hydrolase (CU-NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU-NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU-NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU-NH are identified. Regardless of the wild-type or mutant CU-NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU-NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.
Asunto(s)
Escherichia coli/enzimología , Simulación de Dinámica Molecular , N-Glicosil Hidrolasas/metabolismo , Calcio/química , Dominio Catalítico , Citidina/química , Enlace de Hidrógeno , Hipoxantina/química , Inosina/química , Cinética , Mutación , N-Glicosil Hidrolasas/genética , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Uridina/químicaRESUMEN
This study investigated the effect of different levels of corn supplementation as energy source into palm kernel cake-urea-treated rice straw basal diet on urinary excretion of purine derivatives, nitrogen utilization, rumen fermentation, and rumen microorganism populations. Twenty-seven Dorper lambs were randomly assigned to three treatment groups and kept in individual pens for a 120-day period. The animals were subjected to the dietary treatments as follows: T1: 75.3% PKC + 0% corn, T2: 70.3% PKC + 5% corn, and T3: 65.3% PKC + 10% corn. Hypoxanthine and uric acid excretion level were recorded similarly in lambs supplemented with corn. The microbial N yield and butyrate level was higher in corn-supplemented group, but fecal N excretion, T3 has the lowest level than other groups. Lambs fed T3 had a greater rumen protozoa population while the number of R. flavefaciens was recorded highest in T2. No significant differences were observed for total bacteria, F. succinogenes, R. albus, and methanogen population among all treatment. Based on these results, T3 could be fed to lambs without deleterious effect on the VFA and N balance.
Asunto(s)
Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Dieta/veterinaria , Oryza , Rumen/metabolismo , Zea mays , Animales , Bacterias , Peso Corporal , Suplementos Dietéticos , Fermentación , Concentración de Iones de Hidrógeno , Hipoxantina/química , Nitrógeno/química , Purinas , Ovinos , Oveja Doméstica , Urea/metabolismo , Ácido Úrico/químicaRESUMEN
Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13â mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51â Å resolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6122, with unit-cell parameters a = b = 120.370, c = 238.971â Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by â¼180° relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.