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1.
Adv Exp Med Biol ; 1232: 375-381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893434

RESUMO

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50 µm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750 nm, blue emission filter; FAD channel: excitation 860 nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.


Assuntos
Tecnologia Biomédica , NAD , Imagem Óptica , Animais , Tecnologia Biomédica/instrumentação , Tecnologia Biomédica/métodos , Estudos de Viabilidade , Flavina-Adenina Dinucleotídeo , Xenoenxertos/diagnóstico por imagem , Camundongos , Oxirredução , Fótons
2.
J Chem Phys ; 151(22): 225101, 2019 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-31837685

RESUMO

The ability of night-migratory songbirds to sense the direction of the Earth's magnetic field is increasingly attributed to a photochemical mechanism in which the magnetic field acts on transient radical pairs in cryptochrome flavoproteins located in the birds' eyes. The magnetically sensitive species is commonly assumed to be [FAD•- TrpH•+], formed by sequential light-induced intraprotein electron transfers from a chain of tryptophan residues to the flavin adenine dinucleotide chromophore. However, some evidence points to superoxide, O2 •-, as an alternative partner for the flavin radical. The absence of hyperfine interactions in O2 •- could lead to a more sensitive magnetic compass, but only if the electron spin relaxation of the O2 •- radical is much slower than normally expected for a small mobile radical with an orbitally degenerate electronic ground state. In this study we use spin dynamics simulations to model the sensitivity of a flavin-superoxide radical pair to the direction of a 50 µT magnetic field. By varying parameters that characterize the local environment and molecular dynamics of the radicals, we identify the highly restrictive conditions under which a O2 •--containing radical pair could form the basis of a geomagnetic compass sensor. We conclude that the involvement of superoxide in compass magnetoreception must remain highly speculative until further experimental evidence is forthcoming.


Assuntos
Criptocromos/química , Flavina-Adenina Dinucleotídeo/química , Superóxidos/química , Campos Magnéticos , Modelos Químicos , Simulação de Dinâmica Molecular
3.
Acta Crystallogr D Struct Biol ; 75(Pt 9): 841-851, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478907

RESUMO

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe-4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound ß-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.


Assuntos
Burkholderia cepacia/enzimologia , Glucose Desidrogenase/química , Domínio Catalítico , Cristalografia por Raios X/métodos , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Modelos Moleculares , Proteínas Recombinantes/química
4.
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
5.
Org Biomol Chem ; 17(34): 7973-7984, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31407761

RESUMO

d-Amino acid oxidase (DAAO) is a flavoenzyme whose inhibition is expected to have therapeutic potential in schizophrenia. DAAO catalyses hydride transfer from the substrate to the flavin in the reductive half-reaction, and the flavin is reoxidized by O2 in the oxidative half-reaction. Quantum mechanical/molecular mechanical calculations were performed and their results together with available experimental information were used to elucidate the detailed mechanism of the oxidative half-reaction. The reaction starts with a single electron transfer from FAD to O2, followed by triplet-singlet transition. FAD oxidation is completed by a proton coupled electron transfer to the oxygen species and the reaction terminates with H2O2 formation by proton transfer from the oxidized substrate to the oxygen species via a chain of water molecules. The substrate plays a double role by facilitating the first electron transfer and by providing a proton in the last step. The mechanism differs from the oxidative half-reaction of other oxidases.


Assuntos
D-Aminoácido Oxidase/química , Flavina-Adenina Dinucleotídeo/química , Basidiomycota/enzimologia , Teoria da Densidade Funcional , Humanos , Modelos Químicos , Oxirredução , Oxigênio/química
6.
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 , Ligações de Hidrogênio , Modelos Moleculares , Estrutura Molecular , NAD/química , NADH Desidrogenase/química , Oxirredução , Ligação Proteica , Relação Estrutura-Atividade , Termodinâmica
7.
Phys Chem Chem Phys ; 21(31): 17072-17081, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31313765

RESUMO

The repair of sun-induced DNA lesions by photolyases is driven by a photoinduced electron transfer from a fully reduced FAD to the damaged DNA. A chain of several aromatic residues connecting FAD to solvent ensures the prior photoreduction of the FAD cofactor. In PhrA, a class III CPD photolyase, two branching tryptophan charge transfer pathways have been characterized. According to previous experiments, both pathways play a role in the FAD photoreduction. To provide a molecular insight to the charge transfer abilities of both pathways, we perform multiscales simulations where the protein motion and the positive charge are simultaneously propagated. Our computational approach reveals that one pathway drives a very fast charge transfer whereas the other pathway provides a very good thermodynamic stabilization of the positive charge. During the simulations, the positive charge firstly moves on the fast triad, while a reorganization of the close FAD˙- environment occurs. Then, backward transfers can lead to the propagation of the positive charge on the second pathway. After one nanosecond, we observe a nearly equal probability to find the charge at ending tryptophan of either pathway; eventually the charge distribution will likely evolve towards a charge stabilization on the last tryptophan of the slowest pathway. Our results highlight the role the protein environment, which manages the association of a kinetic and a thermodynamic pathways to trigger a fast and efficient FAD photoreduction.


Assuntos
Reparo do DNA , Desoxirribodipirimidina Fotoliase/química , Modelos Moleculares , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Cinética , Oxirredução , Processos Fotoquímicos , Conformação Proteica , Termodinâmica , Triptofano/química
8.
Photochem Photobiol Sci ; 18(10): 2363-2373, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31290528

RESUMO

Sunlight is a ubiquitous environmental stimulus for the great majority of living organisms on Earth; therefore it is logical to expect the development of "seeing mechanisms" which lead them to successfully adapt to particular ecological niches. Although these mechanisms were recognized in photosynthetic organisms, it was not until recent years that the scientific community found out about light perception in chemotrophic ones. In this review we summarize the current knowledge about the mechanism of light sensing through the blue light receptor BlsA in Acinetobacter baumannii. We highlight its function as a global regulator that pleiotropically modulates a large number of physiological processes, many of which are linked to the ability of this opportunist pathogen to persist in adverse intrahospital environments. Moreover, we describe with some specific examples the molecular basis of how this photoregulator senses blue light and translates this physical signal by modulating gene expression of target regulons. Finally, we discuss the possible course of these investigations needed to dissect this complex regulatory network, which ultimately will help us better understand the A. baumannii physiology.


Assuntos
Acinetobacter baumannii/fisiologia , Proteínas de Bactérias/metabolismo , Luz , Transdução de Sinais/efeitos da radiação , Acetoína/metabolismo , Acinetobacter baumannii/efeitos da radiação , Flavina-Adenina Dinucleotídeo/sangue , Temperatura Ambiente , Virulência
9.
Talanta ; 204: 424-430, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357315

RESUMO

It is very meaningful and useful to select specific aptamers with capacity to distinguish small structural analogues, but it is difficult to carry out by traditional affinity chromatography-SELEX (systematic evolution of ligands by exponential enrichment) based on immobilized target molecules. In this paper, as a proof of concept, we selected DNA aptamers that can specifically recognize and differentiate riboflavin and its derivative flavin adenine dinucleotide (FAD) by a modified method. Here, the random DNA library was indirectly immobilized on streptavidin functional agarose beads by hybridization with its biotinylated short complementary strand, and the specific affinity between aptamers and its target would induce the aptamers to release from beads. Binding specificity can be tailored by performing an additional negative SELEX with the structure analogue of target. After about 10 rounds of selection, 6 aptamers for riboflavin and 2 aptamers for FAD with good affinities were isolated, and their dissociation constants (Kds) were all at low micromolar level. Moreover, as expected, most of these aptamers show high affinity and excellent selectivity for target molecules, almost no binding to structure analogues and purines, indicating this simple method could be used to select specific aptamers to distinguish small molecular targets with similar structures.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA de Cadeia Simples/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Riboflavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Fluorescência , Estudo de Prova de Conceito , Riboflavina/química , Técnica de Seleção de Aptâmeros/métodos
10.
Cell Mol Life Sci ; 76(20): 4023-4042, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31236625

RESUMO

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.


Assuntos
Mitocôndrias/enzimologia , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Subunidades Proteicas/química , Proteínas/genética , Succinato Desidrogenase/química , Animais , Ciclo do Ácido Cítrico/genética , Coenzimas/química , Coenzimas/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fosforilação Oxidativa , Plantas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
11.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 450-454, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204692

RESUMO

The thymidylate synthases ThyA and Thy1 are enzymes that catalyse the formation of thymidine monophosphate from 2'-deoxyuridine monophosphate. Thy1 (or ThyX) requires flavin for catalytic reactions, while ThyA does not. In the present study, the crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus HB8 (TtThy1, TTHA1096) was determined in complex with FAD and phosphate at 2.5 Šresolution. TtThy1 is a tetrameric molecule like other Thy1 proteins, to which four FAD molecules are bound. In the crystal of TtThy1, two phosphate ions were bound to each dUMP-binding site. The characteristic feature of TtThy1 is the existence of an extra C-terminal domain (CTD) consisting of three α-helices and a ß-strand. The function of the CTD is unknown and database analysis showed that this CTD is only shared by part of the Deinococcus-Thermus phylum.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Thermus thermophilus/enzimologia , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/química , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Homologia de Sequência
12.
Eur Biophys J ; 48(4): 395-403, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31053922

RESUMO

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Conformação Molecular , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Espectrometria de Fluorescência , Fatores de Tempo
13.
Chemphyschem ; 20(14): 1793-1798, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31081986

RESUMO

Glucose oxidase is a flavoprotein that is relatively well-studied as a physico-chemical model system. The flavin cofactor is surrounded by several aromatic acid residues that can act as direct and indirect electron donors to photoexcited flavin. Yet, the identity of the photochemical product states is not well established. We present a detailed full spectral reinvestigation of this issue using femtosecond fluorescence and absorption spectroscopy. Based on a recent characterization of the unstable tyrosine cation radical TyrOH•+ , we now propose that the primary photoproduct involves this species, which was previously not considered. Formation of this product is followed by competing charge recombination and radical pair stabilization reactions that involve proton transfer and radical transfer to tryptophan. A minimal kinetic model is proposed, including a fraction of TyrOH.+ that is stabilized up to the tens of picoseconds timescale, suggesting a potential role of this species as intermediate in biochemical electron transfer reactions.


Assuntos
Radicais Livres/química , Glucose Oxidase/química , Glucose Oxidase/efeitos da radiação , Aspergillus niger/enzimologia , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/efeitos da radiação , Proteínas Fúngicas/química , Proteínas Fúngicas/efeitos da radiação , Cinética , Luz , Fotoquímica/métodos , Espectrometria de Fluorescência/métodos , Tirosina/química
14.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 663-676, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31091472

RESUMO

NAD(P)H quinone oxidoreductase 1 (NQO1) is a multi-functional protein that catalyses the reduction of quinones (and other molecules), thus playing roles in xenobiotic detoxification and redox balance, and also has roles in stabilising apoptosis regulators such as p53. The structure and enzymology of NQO1 is well-characterised, showing a substituted enzyme mechanism in which NAD(P)H binds first and reduces an FAD cofactor in the active site, assisted by a charge relay system involving Tyr-155 and His-161. Protein dynamics play important role in physio-pathological aspects of this protein. NQO1 is a good target to treat cancer due to its overexpression in cancer cells. A polymorphic form of NQO1 (p.P187S) is associated with increased cancer risk and certain neurological disorders (such as multiple sclerosis and Alzheimer´s disease), possibly due to its roles in the antioxidant defence. p.P187S has greatly reduced FAD affinity and stability, due to destabilization of the flavin binding site and the C-terminal domain, which leading to reduced activity and enhanced degradation. Suppressor mutations partially restore the activity of p.P187S by local stabilization of these regions, and showing long-range allosteric communication within the protein. Consequently, the correction of NQO1 misfolding by pharmacological chaperones is a viable strategy, which may be useful to treat cancer and some neurological conditions, targeting structural spots linked to specific disease-mechanisms. Thus, NQO1 emerges as a good model to investigate loss of function mechanisms in genetic diseases as well as to improve strategies to discriminate between neutral and pathogenic variants in genome-wide sequencing studies.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Chaperonas Moleculares/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neoplasias/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Animais , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Polimorfismo Genético , Domínios Proteicos , Dobramento de Proteína/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Phys Chem Chem Phys ; 21(22): 11642-11650, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116217

RESUMO

Redox reactions play a key role in various biological processes, including photosynthesis and respiration. Quantitative and predictive computational characterization of redox events is therefore highly desirable for enriching our knowledge on mechanistic features of biological redox-active macromolecules. Here, we present a computational protocol exploiting polarizable embedding hybrid quantum-classical approach and resulting in accurate estimates of redox potentials of biological macromolecules. A special attention is paid to fundamental aspects of the theoretical description such as the effects of environment polarization and of the long-range electrostatic interactions on the computed energetic parameters. Environment (protein and the solvent) polarization is shown to be crucial for accurate estimates of the redox potential: hybrid quantum-classical results with and without account for environment polarization differ by 1.4 V. Long-range electrostatic interactions are shown to contribute significantly to the computed redox potential value even at the distances far beyond the protein outer surface. The approach is tested on simulating reduction potential of cryptochrome 1 protein from Arabidopsis thaliana. The theoretical estimate (0.07 V) of the midpoint reduction potential is in good agreement with available experimental data (-0.15 V).


Assuntos
Proteínas de Arabidopsis/química , Criptocromos/química , Flavina-Adenina Dinucleotídeo/química , Arabidopsis/química , Teoria da Densidade Funcional , Modelos Químicos , Oxirredução , Eletricidade Estática
16.
Phys Chem Chem Phys ; 21(22): 11956-11966, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31134233

RESUMO

Photolyases (PL) and cryptochromes (CRY) are light-sensitive flavoproteins, respectively, involved in DNA repair and signal transduction. Their activation is triggered by an electron transfer process, which partially or fully reduces the photo-activated FAD cofactor. The full reduction additionally requires a proton transfer to the isoalloxazine ring. In plant CRY, an efficient proton transfer takes place within several µs, enabled by a conserved aspartate working as a proton donor, whereas in E. coli PL a proton transfer occurs in the 4 s timescale without any obvious proton donor, indicating the presence of a long-range proton transfer pathway. Unexpectedly, the insertion of an aspartate as a proton donor in a suitable position for proton transfer in E. coli PL does not initiate a transfer process similar to plant CRY, but even prevents the formation of a protonated FAD. In the present work, thanks to a combination of classical molecular dynamics and state-of-the-art DFTB3/MM simulations, we identify a proton transfer pathway from bulk to FAD in E. coli PL associated with a free energy profile in agreement with the experimental kinetics data. The free energy profiles of the proton transfer between aspartate and FAD show an inversion of the driving force between plant CRY and E. coli PL mutants. In the latter, the proton transfer from the aspartate is faster than in plant CRY but also thermodynamically disfavoured, in agreement with the experimental data. Our results further illustrate the fine tuning of the electrostatic FAD environment and the adaptability of the FAD pocket to ensure the divergent functions of the members of the PL-CRY family.


Assuntos
Criptocromos/química , Desoxirribodipirimidina Fotoliase/química , Flavina-Adenina Dinucleotídeo/química , Prótons , Sítios de Ligação , Teoria da Densidade Funcional , Desoxirribodipirimidina Fotoliase/genética , Escherichia coli/química , Modelos Químicos , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Termodinâmica , Água/química
17.
J Agric Food Chem ; 67(23): 6532-6540, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31099250

RESUMO

In this work, modular engineering of Escherichia coli was peformed to improve flavin production and the conversion ratio of riboflavin (RF) to FMN/FAD. The RF operon and the bifunctional RF kinase/FAD synthetase were divided into two separate modules. The two modules were expressed at different levels to produce RF: ribF ratios ranging from 2:20 to 7:5. The best strain respectively produced 324.1 and 171.6 mg/L of FAD and FMN in shake flask fermentation, and the titers reached 1899.3 and 872.7 mg/L in a fed-batch process. Furthermore, error-prone PCR (epPCR) of the E. coli ribF gene was performed. The highest FMN production of the best mutant reached 586.1 mg/L in shake flask cultivation. Moreover, this mutant produced 1017.5 mg/L FMN with a greatly reduced proportion of FAD in fermenter culture. To the best of our knowledge, this is the highest production of FAD and FMN in a microbial fermentation process reported to date.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/biossíntese , Flavina-Adenina Dinucleotídeo/biossíntese , Técnicas de Cultura Celular por Lotes , Fermentação , Engenharia Metabólica
18.
Bioelectrochemistry ; 128: 66-73, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30928867

RESUMO

Different carbon-based nanostructures were used to investigate direct electron transfer (DET) of TetX2 monooxygenase (TetX2), and an enzyme-based biosensor for sensitive determination of tetracycline (TC) also fabricated. A polyethyleneimine (PEI) with positive charge groups was used for immobilization of TetX2 on modified glassy carbon electrodes. Cyclic voltammetry (CV) was employed to study the electrochemical characteristics of the immobilized enzyme and the performance of the proposed biosensor. Amongst multiple carbon-modified electrodes, nano-porous glassy carbon electrode (NPGCE) was selected because of its amplified signal response for flavin adenine dinucleotide (FAD) and superior electrocatalytic behavior toward oxygen reduction. The cyclic voltammogram of PEI/TetX2/NPGCE showed two couple of well-defined and quasi-reversible redox peaks of FAD, consistent with the realization of DET. The prepared electrode was then successfully introduced as a biosensing interface based on the oxygen reduction peak current, resulting in a linear range response from 0.5 to 5 µM with a good detection limit of 18 nM. The as-fabricated electrode demonstrates a fast response and excellent stability for the detection of TC. The results indicate that this simple, rapid, eco-friendly and economic strategy of PEI/TetX2/NPGCE preparation has potential for the fabrication of an enzyme-based biosensor for the practical detection of TC in food products.


Assuntos
Técnicas Biossensoriais , Carbono , Resíduos de Drogas/análise , Técnicas Eletroquímicas/instrumentação , Eletrodos , Enzimas Imobilizadas/metabolismo , Oxigenases de Função Mista/metabolismo , Tetraciclina/análise , Catálise , Flavina-Adenina Dinucleotídeo/análise , Oxirredução , Polietilenoimina/química , Reprodutibilidade dos Testes
19.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959771

RESUMO

Mitochondrial dysfunction is a core feature of acute pancreatitis, a severe disease in which oxidative stress is elevated. Mitochondrial targeting of antioxidants is a potential therapeutic strategy for this and other diseases, although thus far mixed results have been reported. We investigated the effects of mitochondrial targeting with the antioxidant MitoQ on pancreatic acinar cell bioenergetics, adenosine triphosphate (ATP) production and cell fate, in comparison with the non-antioxidant control decyltriphenylphosphonium bromide (DecylTPP) and general antioxidant N-acetylcysteine (NAC). MitoQ (µM range) and NAC (mM range) caused sustained elevations of basal respiration and the inhibition of spare respiratory capacity, which was attributable to an antioxidant action since these effects were minimal with DecylTPP. Although MitoQ but not DecylTPP decreased cellular NADH levels, mitochondrial ATP turnover capacity and cellular ATP concentrations were markedly reduced by both MitoQ and DecylTPP, indicating a non-specific effect of mitochondrial targeting. All three compounds were associated with a compensatory elevation of glycolysis and concentration-dependent increases in acinar cell apoptosis and necrosis. These data suggest that reactive oxygen species (ROS) contribute a significant negative feedback control of basal cellular metabolism. Mitochondrial targeting using positively charged molecules that insert into the inner mitochondrial member appears to be deleterious in pancreatic acinar cells, as does an antioxidant strategy for the treatment of acute pancreatitis.


Assuntos
Células Acinares/metabolismo , Antioxidantes/metabolismo , Linhagem da Célula , Metabolismo Energético , Mitocôndrias/metabolismo , Pâncreas/citologia , Acetilcisteína/farmacologia , Células Acinares/efeitos dos fármacos , Trifosfato de Adenosina/biossíntese , Animais , Morte Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Flavina-Adenina Dinucleotídeo/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , NAD/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Oxirredução , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
20.
Ann Biomed Eng ; 47(7): 1564-1574, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30963380

RESUMO

The kidney is one of the most radiosensitive organs; it is the primary dose-limiting organ in radiotherapies for upper abdominal cancers. The role of mitochondrial redox state in the development and treatment of renal radiation injury, however, remains ill-defined. This study utilizes 3D optical cryo-imaging to quantify renal mitochondrial bioenergetics dysfunction after 13 Gy leg-out partial body irradiation (PBI). Furthermore, the mitigating effects of lisinopril (lisino), an anti-hypertensive angiotensin converting enzyme inhibitor, is assessed in renal radiation-induced injuries. Around day 150 post-irradiation, kidneys are harvested for cryo-imaging. The 3D images of the metabolic indices (NADH, nicotinamide adenine dinucleotide, and FAD, flavin adenine dinucleotide) are acquired, and the mitochondrial redox states of the irradiated and irradiated + lisino kidneys are quantified by calculating the volumetric mean redox ratio (NADH/FAD). PBI oxidized renal mitochondrial redox state by 78%. The kidneys from the irradiated + lisino rats showed mitigation of mitochondrial redox state by 93% compared to the PBI group. The study provides evidence for an altered bioenergetics and energy metabolism in the rat model of irradiation-induced kidney damage. In addition, the results suggest that lisinopril mitigates irradiation damage by attenuating the oxidation of mitochondria leading to increase redox ratio.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Nefropatias/tratamento farmacológico , Rim/efeitos da radiação , Lisinopril/uso terapêutico , Mitocôndrias/efeitos da radiação , Lesões por Radiação/tratamento farmacológico , Animais , Feminino , Flavina-Adenina Dinucleotídeo/metabolismo , Raios gama , Imagem Tridimensional , Rim/metabolismo , Nefropatias/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Lesões por Radiação/metabolismo , Ratos
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