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1.
Int J Nanomedicine ; 15: 275-300, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021180

RESUMO

Gold nanoparticles (AuNPs) are extensively studied nanoparticles (NPs) and are known to have profound applications in medicine. There are various methods to synthesize AuNPs which are generally categorized into two main types: chemical and physical synthesis. Continuous efforts have been devoted to search for other more environmental-friendly and economical large-scale methods, such as environmentally friendly biological methods known as green synthesis. Green synthesis is especially important to minimize the harmful chemical and toxic by-products during the conventional synthesis of AuNPs. Green materials such as plants, fungi, microorganisms, enzymes and biopolymers are currently used to synthesize various NPs. Biosynthesized AuNPs are generally safer for use in biomedical applications since they come from natural materials themselves. Multiple surface functionalities of AuNPs allow them to be more robust and flexible when combined with different biological assemblies or modifications for enhanced applications. This review focuses on recent developments of green synthesized AuNPs and discusses their numerous biomedical applications. Sources of green materials with successful examples and other key parameters that determine the functionalities of AuNPs are also discussed in this review.


Assuntos
Ouro/química , Química Verde/métodos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Animais , Bactérias/química , Sistemas de Liberação de Medicamentos , Fungos/química , Humanos , NAD/química , Fenóis/química , Plantas/química , Proteínas/química , Terpenos/química
2.
Chem Commun (Camb) ; 56(18): 2723-2726, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32021996

RESUMO

Encapsulation of two enzymes, alcohol dehydrogenase (ADH) and glucose oxidase (GOx), within peroxidase-like tourmaline microparticle (TM)-based colloidosomes was used to construct a functionalized microsystem capable of sustainable cascade cycling of nicotinamide cofactor (NAD+/NADH) via chemical signaling between spatially confined dual-enzyme and active membranes.


Assuntos
Álcool Desidrogenase/metabolismo , Glucose Oxidase/metabolismo , NAD/metabolismo , Álcool Desidrogenase/química , Glucose Oxidase/química , NAD/química , Tamanho da Partícula , Propriedades de Superfície
3.
J Agric Food Chem ; 68(7): 2139-2145, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31973519

RESUMO

α-Pinene is an important monoterpene that is widely used as a pharmaceutical product, biofuel, and so forth. We first established a cell-free system with modular cocatalysis for the production of pinene from glucose. After optimization of the compositions of the cell-free reaction mixture using the Plackett-Burman experimental design and the path of steepest ascent, the production of pinene increased by 57%. It was found that ammonium acetate, NAD+, and NADPH are the three most important parameters for the production of pinene. Mix-and-match experiments showed that the simultaneous addition of the lysate of Escherichia coli overexpressing native 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, SufBCD Fe-S cluster assembly protein, isopentenyl-diphosphate isomerase, and Pinus taeda pinene synthase improved the production of pinene. Increasing the enzyme concentration of the extract further enhanced the production of pinene to 1256.31 ± 46.12 mg/L with a productivity of 104.7 mg/L h, almost 1.2-fold faster than any system reported thus far. This study demonstrates that a cell-free system is a powerful and robust platform for biomanufacture.


Assuntos
Monoterpenos Bicíclicos/química , Escherichia coli/química , Monoterpenos Bicíclicos/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo
4.
Nat Chem Biol ; 16(1): 87-94, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31768035

RESUMO

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.


Assuntos
NADP/química , Mononucleotídeo de Nicotinamida/química , Oxirredução , Biocatálise , Carbono/química , Cromatografia Gasosa , Cicloexanonas/química , Escherichia coli/metabolismo , Cinética , NAD/química , Mononucleotídeo de Nicotinamida/genética , Conformação Proteica , Engenharia de Proteínas , Pseudomonas putida/metabolismo , Ralstonia/metabolismo , Software
5.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1107-1118, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793904

RESUMO

The core of ß-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. ß-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD+-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD+, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD+ binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.


Assuntos
Ácido 2-Aminoadípico/química , Proteínas de Bactérias/química , NAD/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Ácidos Picolínicos/química , Streptomyces/metabolismo , Domínio Catalítico , Especificidade por Substrato
6.
ACS Appl Mater Interfaces ; 11(50): 47625-47634, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31794177

RESUMO

DNA release from an electrode surface was stimulated by application of a mild electrical potential (0 V vs Ag/AgCl). The release process was activated by interfacial pH increase originating from H+ consumption during O2 reduction bio-electrocatalyzed by bilirubin oxidase immobilized at the electrode surface. The pH increase resulted in a change of the electrical charge from positive to negative at the surface of SiO2 nanoparticles (200 nm) associated with the electrode surface and functionalized with trigonelline and boronic acid. While the negatively charged DNA molecules were electrostatically bound to the positively charged surface, the negative charge produced upon O2 reduction resulted in the DNA repulsion and release from the modified interface. The small electrical potential for O2 reduction resulting in the interface recharge was allowed due to the bio-electrocatalysis using bilirubin oxidase enzyme. While, in the first set of experiments, the potential was applied on the modified electrode from an electrochemical instrument, later it was generated in situ by biocatalytic or photo-biocatalytic processes at a connected electrode. A multistep biocatalytic cascade generating NADH or photosynthetic process in thylakoid membranes was used to produce in situ a small potential to stimulate the DNA release catalyzed by bilirubin oxidase. The designed system can be used for different release processes triggered by various signals (electrical, biomolecular, and light signals, etc.), thus representing a general interfacial platform for the controlled release of different biomolecules and nanosize species.


Assuntos
Técnicas Biossensoriais , Enzimas Imobilizadas/química , NAD/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Biocatálise , DNA/química , Eletrodos , Hidrogênio/química , Concentração de Íons de Hidrogênio , NAD/química , Nanopartículas/química , Oxigênio/química , Propriedades de Superfície , Tilacoides/química
7.
Sensors (Basel) ; 20(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861836

RESUMO

Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is overexpressed in cancer cells and associated with the drug resistance factor of cancer. The objective of this work is the development of fluorescent probes for the efficient detection of hNQO1 activity in cancer cells, which can be employed for the cancer diagnosis and therapeutic agent development. Herein, we report naphthalimide-based fluorescent probes 1 and 2 that can detect hNQO1. For hNQO1 activity, the probes showed a significant fluorescence increase at 540 nm. In addition, probe 1, the naphthalimide containing a triphenylphosphonium salt, showed an enhanced enzyme efficiency and rapid detection under a physiological condition. The detection ability of probe 1 was superior to that of other previously reported probes. Moreover, probe 1 was less cytotoxic during the cancer cell imaging and readily provided a strong fluorescence in hNQO1-overexpressed cancer cells (A549). We proposed that probe 1 can be used to detect hNQO1 expression in live cells and it will be applied to develop the diagnosis and customized treatment of hNQO1-related disease.


Assuntos
Materiais Biocompatíveis/metabolismo , Corantes Fluorescentes/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftalimidas/metabolismo , Células A549 , Materiais Biocompatíveis/química , Linhagem Celular Tumoral , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , NAD/química , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , Naftalimidas/química , Espectrometria de Fluorescência
8.
Biochemistry (Mosc) ; 84(11): 1403-1410, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760926

RESUMO

Genomes of photoautotrophic organisms containing type I photosynthetic reaction center were searched for the rnf genes encoding Na+-translocating ferredoxin:NAD+ oxidoreductase (RNF). These genes were absent in heliobacteria, cyanobacteria, algae, and plants; however, genomes of many green sulfur bacteria (especially marine ones) were found to contain the full rnf operon. Analysis of RNA isolated from the marine green sulfur bacterium Chlorobium phaeovibrioides revealed a high level of rnf expression. It was found that the activity of Na+-dependent flavodoxin:NAD+ oxidoreductase detected in the membrane fraction of Chl. phaeovibrioides was absent in the membrane fraction of the freshwater green sulfur bacterium Chlorobaculum limnaeum, which is closely related to Chl. phaeovibrioides but whose genome lacks the rnf genes. Illumination of the membrane fraction of Chl. phaeovibrioides but not of Cba. limnaeum resulted in the light-induced NAD+ reduction. Based on the obtained data, we concluded that in some green sulfur bacteria, RNF may be involved in the NADH formation that should increase the efficiency of light energy conservation in these microorganisms and can serve as the first example of the use of Na+ energetics in photosynthetic electron transport chains.


Assuntos
Proteínas de Bactérias/metabolismo , Chlorobi/metabolismo , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte de Elétrons , Luz , NAD/química , NAD/metabolismo , Oxirredutases/química , Oxirredutases/genética , Fotossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
9.
Molecules ; 24(21)2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31731395

RESUMO

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.


Assuntos
Androstadienos/metabolismo , Bacillus subtilis , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Esteroides/biossíntese , Androstadienos/química , Androstadienos/farmacologia , Bacillus subtilis/química , Catalase/química , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Microambiente Celular , Peróxido de Hidrogênio/química , Engenharia Metabólica , Complexos Multienzimáticos/química , Mycobacteriaceae/genética , Mycobacteriaceae/metabolismo , NAD/química , NAD/metabolismo , NADH NADPH Oxirredutases/química , Espécies Reativas de Oxigênio/metabolismo , Esteroides/metabolismo , Esteróis/metabolismo
10.
J Agric Food Chem ; 67(48): 13327-13332, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31715101

RESUMO

The biochemical basis of lower metmyoglobin reducing activity (MRA) in high-oxygen modified atmospheric packaged (HiOx-MAP) beef than those in vacuum and polyvinyl chloride (PVC) packaging is not clear. To explore this, the effects of lipid oxidation products with varying carbon chain length on lactate dehydrogenase (LDH) and NADH-dependent metmyoglobin reductase activity were evaluated. Surface color, MRA, and lipid oxidation of beef longissimus lumborum steaks (n = 10) were measured during 6-day display. Further, two enzymes, LDH and NADH-dependent metmyoglobin reductase (n = 5), critical for MRA were incubated with or without (control) lipid oxidation products of varying carbon chain length: malondialdehyde (3-carbon), hexenal (6-carbon), and 4-hydroxynonenal (9-carbon). Steaks in HiOx-MAP had greater (P < 0.05) redness than vacuum and PVC, but had lower (P < 0.05) MRA and greater (P < 0.05) lipid oxidation on day 6. LDH and NADH-dependent metmyoglobin reductase activities were differentially influenced by lipid oxidation products (P < 0.05). The results indicate that the difference in reactivity of various lipid oxidation products on LDH (HNE > MDA = hexenal) and NADH-dependent metmyoglobin reductase (HNE = MDA > hexenal) activity could be responsible for lower MRA in HiOx-MAP.


Assuntos
Carbono/química , L-Lactato Desidrogenase/química , Lipídeos/química , NADH NADPH Oxirredutases/química , Carne Vermelha/análise , Animais , Carbono/metabolismo , Bovinos , Embalagem de Alimentos , L-Lactato Desidrogenase/metabolismo , Metamioglobina/química , Metamioglobina/metabolismo , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , NAD/química , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução
11.
Chem Soc Rev ; 48(23): 5596-5615, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31675020

RESUMO

A number of self-sufficient hydride transfer processes have been reported in biocatalysis, with a common feature being the dependence on nicotinamide as a cofactor. This cofactor is provided in catalytic amounts and serves as a hydride shuttle to connect two or more enzymatic redox events, usually ensuring overall redox neutrality. Creative systems were designed to produce synthetic sequences characterized by high hydride economy, typically going in hand with excellent atom economy. Several redox enzymes have been successfully combined in one-pot one-step to allow functionalization of a large variety of molecules while preventing by-product formation. This review analyzes and classifies the various strategies, with a strong focus on efficiency, which is evaluated here in terms of the hydride economy and measured by the turnover number of the nicotinamide cofactor(s). The review ends with a critical evaluation of the reported systems and highlights areas where further improvements might be desirable.


Assuntos
Enzimas/metabolismo , NAD/metabolismo , Biocatálise , Enzimas/química , Isomerismo , NAD/química , Oxirredução , Especificidade por Substrato
12.
Anal Chim Acta ; 1091: 95-102, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31679579

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1) activity is closely related to tumor, which is a promising biomarker for cancer diagnosis. So far, only a few methods have been developed for PARP-1 activity assay because both PARP-1 and its catalytic products lack valuable optical or electrochemical property. Herein, we propose a more specific method to label probes on great deal of phosphate groups of PAR. Firstly, versatile peptides were used to prepare CuNPs. This peptide not only worked as reducing agent to prepare CuNPs but also had guanidine groups to label PAR autonomously and specifically. Unlike most previously reported methods based on unspecific electrostatic interactions, CuNPs probes covered by guanidine groups labelled PAR with phosphate groups via intense covalent-like interactions. On the other hand, PARP-1 catalyzed the formation of PAR in each isolated reaction container of the detection array, realizing the high-throughput detection and enhancing the detection efficiency. Ultimately, CuNPs were oxidized into Cu2+ and precisely detected by stripping voltammetry. Hence, selectivity and efficiency of PARP-1 detection were both improved. Meanwhile, this approach was successfully used to detect the efficiency of PARP-1 inhibitor and the PARP-1 contents in real cells, indicating its great potential for clinical diagnosis and high-throughput PARP-1 inhibitor screen.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ensaios Enzimáticos/métodos , Nanopartículas Metálicas/química , Poli(ADP-Ribose) Polimerase-1/sangue , Linhagem Celular Tumoral , Cobre/química , DNA/química , Humanos , Limite de Detecção , NAD/química , Ácido Nítrico/química , Peptídeos/química , Poli(ADP-Ribose) Polimerase-1/química
13.
Molecules ; 24(22)2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31752261

RESUMO

Nicotinamide adenine dinucleotide (NAD) serves as an essential redox co-factor and mediator of multiple biological processes. Besides its well-established role in electron transfer reactions, NAD serves as a substrate for other biotransformations, which, at the molecular level, can be classified as protein post-translational modifications (protein deacylation, mono-, and polyADP-ribosylation) and formation of signaling molecules (e.g., cyclic ADP ribose). These biochemical reactions control many crucial biological processes, such as cellular signaling and recognition, DNA repair and epigenetic modifications, stress response, immune response, aging and senescence, and many others. However, the links between the biological effects and underlying molecular processes are often poorly understood. Moreover, NAD has recently been found to tag the 5'-ends of some cellular RNAs, but the function of these NAD-capped RNAs remains largely unrevealed. Synthetic NAD analogs are invaluable molecular tools to detect, monitor, structurally investigate, and modulate activity of NAD-related enzymes and biological processes in order to aid their deeper understanding. Here, we review the recent advances in the design and development of NAD analogs as probes for various cellular NAD-related enzymes, enzymatic inhibitors with anticancer or antimicrobial therapeutic potential, and other NAD-related chemical biology tools. We focus on research papers published within the last 10 years.


Assuntos
NAD/análogos & derivados , NAD/metabolismo , Oxirredução , Animais , Bioquímica , Química Farmacêutica , Descoberta de Drogas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , NAD/química , NAD/farmacologia , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
14.
Phys Chem Chem Phys ; 21(44): 24572-24583, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663551

RESUMO

In this work, poly(N,N'-dimethylaminoethylmethacrylate-co-N-isopropylacrylamide) copolymer films were polymerized on the surface of Au electrodes with a facile one-step method, and Au nanoclusters (AuNCs) and tetraphenylethene (TPE) were synchronously embedded in the films, designated as P(DMA-co-NIPA)/AuNCs/TPE. Ferrocene dicarboxylic acid (FDA), an electroactive probe in solution displayed inverse pH- and SO42--sensitive on-off cyclic voltammetric (CV) behaviors at the film electrodes. The electrocatalytic oxidation of nicotinamide adenine dinucleotide (NADH) mediated by FDA in solution could substantially amplify the CV response difference between the on and off states. Moreover, the two fluorescence emission (FL) signals from the TPE constituent at 450 nm and AuNCs component at 660 nm in the films also demonstrated SO42-- and pH-sensitive behaviors. Based on the aforementioned results, a 4-input/9-output biomolecular logic circuit was constructed with pH, Na2SO4, FDA and NADH as the inputs, and the CV signals and the FL responses at 450 and 660 nm at different levels as the outputs. Additionally, some functional non-Boolean devices were elaborately designed on an identical platform, including a 1-to-2 decoder, a 2-to-1 encoder, a 1-to-2 demultiplexer and different types of keypad locks. This work combines copolymer films, bioelectrocatalysis, and fluorescence together so that more complicated biocomputing systems could be established. This work may pave a new way to develop advanced and sophisticated biocomputing logic circuits and functional devices in the future.


Assuntos
Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , NAD/química , Polímeros/química , Estilbenos/química , Benzoatos/química , Eletrodos , Compostos Ferrosos/química , Ouro/química , Concentração de Íons de Hidrogênio , Metalocenos , Oxirredução , Espectrometria de Fluorescência , Sulfatos/química
15.
Org Biomol Chem ; 17(38): 8716-8720, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31538639

RESUMO

Nicotinamide adenine dinucleotide, NAD+, is an essential cofactor and substrate for many cellular enzymes. Its sustained intracellular levels have been linked to improved physiological end points in a range of metabolic diseases. Biosynthetic precursors to NAD+ include nicotinic acid, nicotinamide, the ribosylated parents and the phosphorylated form of the ribosylated parents. By combining solvent-assisted mechanochemistry and sealed reaction conditions, access to the ribosylated NAD+ precursors and to the isotopologues of NAD+ precursors was achieved in high yields and levels of purity. The latter is critical as it offers means to better trace biosynthetic pathways to NAD+, investigate the multifaceted roles of the intracellular NAD+ pools, and better exploit NAD+ biology.


Assuntos
NAD/síntese química , Estrutura Molecular , NAD/química
16.
Nanoscale ; 11(41): 19285-19290, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31539009

RESUMO

While a large number of studies deal with biomedical applications of various types of nanoparticles synthesized using wet chemistry, we propose the concept of targeted biosynthesis of nanoparticles in the living brain. Here we demonstrate that the pathological biochemical process of accumulation of reduced pyridine nucleotides under deleterious conditions of brain hypoxia can be redirected to drive the biosynthesis of biocompatible Au nanoparticles from a precursor salt in situ in the immediate vicinity of the hypoxia site, thereby restoring the redox status of the brain.


Assuntos
Encéfalo/metabolismo , Hipóxia Celular , Ouro/química , Nanopartículas Metálicas/química , Animais , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NAD/química , Espalhamento a Baixo Ângulo , Difração de Raios X
18.
Chemistry ; 25(67): 15288-15294, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31483908

RESUMO

Nanoscale assemblies of DNA strands are readily designed and can be generated in a wide range of shapes and sizes. Turning them into solids that bind biomolecules reversibly, so that they can act as active material in flow cells, is a challenge. Among the biomolecular ligands, cofactors are of particular interest because they are often the most expensive reagents of biochemical transformations, for which controlled release and recycling are desirable. We have recently described DNA triplex motifs that bind adenine-containing cofactors, such as NAD, FAD and ATP, reversibly with low micromolar affinity. We sought ways to convert the soluble DNA motifs into a macroporous solid for cofactor binding. While assemblies of linear and branched DNA motifs produced hydrogels with undesirable properties, long DNA triplexes treated with protamine gave materials suitable for flow cells. Using exchangeable cells in a flow system, thermally controlled loading and discharge were demonstrated. Employing a flow cell loaded with ATP, bioluminescence was induced through thermal release of the cofactor. The results show that materials generated from functional DNA structures can be successfully employed in macroscopic devices.


Assuntos
Adenina/química , DNA/química , Nanopartículas/química , Trifosfato de Adenosina/química , Sítios de Ligação , Flavina-Adenina Dinucleotídeo/química , Ligantes , NAD/química , Motivos de Nucleotídeos , Espectrometria de Fluorescência/métodos , Termodinâmica
19.
Biosens Bioelectron ; 143: 111598, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31442753

RESUMO

Herein, we report the anionic surfactant, ethylene diamine tetraacetic acid (EDTA), mediated synthesis of WO3 nanoparticles and its subsequent modification through gamma irradiation (GI) and electrochemical immobilization with nicotinamide adenine dinucleotide (NAD). Glassy carbon electrode (GCE) modified with GI-WO3 NPs and the enzyme NAD exhibited strong electro-oxidation of three important biomolecules such as norepinephrine (NEP), melatonin (MEL) and nicotine (NIC) in 0.1 M phosphate buffer saline (PBS) at physiological pH of 7. Square wave voltammetry (SWV) studies exhibited three well-defined peaks at potentials of 120, 570 and 840 mV, corresponding to the oxidation of NEP, MEL and NIC respectively, indicating that simultaneous determination of these compounds is feasible at the NAD/GI EDTA-WO3/GCE. The proposed sensor displayed a wide linear range of 0.010-1000 µM with the lowest detection limit of 1.4 nM for NEP, 2.6 nM for MEL and 1.7 nM for NIC respectively. Furthermore, the modified electrode was successfully applied to detect NEP, MEL and NIC in pharmaceutical and cigarette samples with excellent selectivity and reproducibility.


Assuntos
Técnicas Biossensoriais , Melatonina/isolamento & purificação , Nicotina/isolamento & purificação , Norepinefrina/isolamento & purificação , Técnicas Eletroquímicas , Limite de Detecção , Melatonina/química , NAD/química , Nanopartículas/química , Nanotubos de Carbono/química , Nicotina/química , Norepinefrina/química , Óxidos/química , Tungstênio/química
20.
Phys Chem Chem Phys ; 21(33): 18105-18118, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31396604

RESUMO

With the emergence of drug-resistant Plasmodium falciparum, the treatment of malaria has become a significant challenge; therefore, the development of antimalarial drugs acting on new targets is extremely urgent. In Plasmodium falciparum, type II nicotinamide adenine dinucleotide (NADH) dehydrogenase (NDH-2) is responsible for catalyzing the transfer of two electrons from NADH to flavin adenine dinucleotide (FAD), which in turn transfers the electrons to coenzyme Q (CoQ). As an entry enzyme for oxidative phosphorylation, NDH-2 has become one of the popular targets for the development of new antimalarial drugs. In this study, reliable motion trajectories of the NDH-2 complex with its co-factors (NADH and FAD) and inhibitor, RYL-552, were obtained by comparative molecular dynamics simulations. The influence of cofactor binding on the global motion of NDH-2 was explored through conformational clustering, principal component analysis and free energy landscape. The molecular interactions of NDH-2 before and after its binding with the inhibitor RYL-552 were analyzed, and the key residues and important hydrogen bonds were also determined. The results show that the association of RYL-552 results in the weakening of intramolecular hydrogen bonds and large allosterism of NDH-2. There was a significant positive correlation between the angular change of the key pocket residues in the NADH-FAD-pockets that represents the global functional motion and the change in distance between NADH-C4 and FAD-N5 that represents the electron transfer efficiency. Finally, the possible non-competitive inhibitory mechanism of RYL-552 was proposed. Specifically, the association of inhibitors with NDH-2 significantly affects the global motion mode of NDH-2, leading to widening of the distance between NADH and FAD through cooperative motion induction; this reduces the electron transfer efficiency of the mitochondrial respiratory chain. The simulation results provide useful theoretical guidance for subsequent antimalarial drug design based on the NDH-2 structure and the respiratory chain electron transfer mechanism.


Assuntos
Antimaláricos/química , Cetonas/química , NADH Desidrogenase/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Quinolinas/química , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , NAD/química , NADH Desidrogenase/química , Oxirredução , Ligação Proteica , Relação Estrutura-Atividade , Termodinâmica
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