RESUMO
Flavin-dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer-Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L-Orn to a class B monooxygenase, the ornithine hydroxylase from Aspergillus $$ Aspergillus $$ fumigatus $$ fumigatus $$ known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from the out to the in position. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr-loops that play critical roles in the process.
Assuntos
Flavina-Adenina Dinucleotídeo , Oxigenases de Função Mista , Oxigenases de Função Mista/química , NADP/química , Oxirredução , Domínio Catalítico , Flavina-Adenina Dinucleotídeo/químicaRESUMO
Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.
Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/metabolismo , Biocatálise , Descarboxilação , Flavina-Adenina Dinucleotídeo/química , Oxigenases de Função Mista/química , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
Flavin cofactors, like flavin adenine dinucleotide (FAD), are important electron shuttles in living systems. They catalyze a wide range of one- or two-electron redox reactions. Experimental investigations include UV-vis as well as infrared spectroscopy. FAD in aqueous solution exhibits a significantly shorter excited state lifetime than its analog, the flavin mononucleotide. This finding is explained by the presence of a "stacked" FAD conformation, in which isoalloxazine and adenine moieties form a π-complex. Stacking of the isoalloxazine and adenine rings should have an influence on the frequency of the vibrational modes. Density functional theory (DFT) studies of the closed form of FAD in microsolvation (explicit water) were used to reproduce the experimental infrared spectra, substantiating the prevalence of the stacked geometry of FAD in aqueous surroundings. It could be shown that the existence of the closed structure in FAD can be narrowed down to the presence of only a single water molecule between the third hydroxyl group (of the ribityl chain) and the N7 in the adenine ring of FAD.
Assuntos
Teoria da Densidade Funcional , Flavina-Adenina Dinucleotídeo/química , Prótons , Espectrofotometria Infravermelho , Transporte de Elétrons , Conformação Molecular , Simulação de Dinâmica Molecular , Água/químicaRESUMO
Graphene oxide-gold nanoparticle (AuNPs@GO) hybrids were fabricated in water dispersions of graphene oxide (GO) and Au precursor completely free of stabilizing agents by UV-light irradiation. Gold nanoparticle (AuNP) nucleation, growth, and stabilization mechanisms at the surface of GO are discussed on the basis of UV-Vis, Raman, IR, and X-Ray photo-spectroscopy studies. The analyses of AuNPs@GO hybrids by transmission electron microscopy (TEM), thermogravimetric (TGA) and electrochemical tests show that they exhibit outstanding chemical, thermal and electrochemical stabilities. Thus, AuNPs@GO biosensing platforms were fabricated for surface enhanced Raman spectroscopy (SERS) detection of crystal violet (CV), a SERS standard molecule, and in a different set of experiments, for flavin adenine dinucleotide (FAD), a flavoprotein coenzyme that plays an important role in many oxidoreductase and reversible redox conversions in biochemical reactions. AuNPs@GO hybrids synthesized by using UV light irradiation show exceptional stability and high intensification of the Raman signals showing that they have high potential for use as biomedical probes for the detection, monitoring, and diagnosis of medical diseases.
Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Grafite/química , Nanopartículas Metálicas/química , Técnicas Eletroquímicas , Flavina-Adenina Dinucleotídeo/química , Microscopia Eletrônica de Transmissão , Oxirredução , Óxidos/química , Oxirredutases/química , Oxirredutases/metabolismo , Espectroscopia Fotoeletrônica , Espectrofotometria , Termogravimetria , Raios Ultravioleta , Água/químicaRESUMO
A combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular modelling methods were used to understand the potent inhibitory NAD(P)H:quinone oxidoreductase 1 (NQO1) activity of a set of 52 heterocyclic quinones. Molecular docking results indicated that some favourable interactions of key amino acid residues at the binding site of NQO1 with these quinones would be responsible for an improvement of the NQO1 activity of these compounds. The main interactions involved are hydrogen bond of the amino group of residue Tyr128, π-stacking interactions with Phe106 and Phe178, and electrostatic interactions with flavin adenine dinucleotide (FADH) cofactor. Three models were prepared by 3D-QSAR analysis. The models derived from Model I and Model III, shown leave-one-out cross-validation correlation coefficients (q2LOO ) of .75 and .73 as well as conventional correlation coefficients (R2 ) of .93 and .95, respectively. In addition, the external predictive abilities of these models were evaluated using a test set, producing the predicted correlation coefficients (r2pred ) of .76 and .74, respectively. The good concordance between the docking results and 3D-QSAR contour maps provides helpful information about a rational modification of new molecules based in quinone scaffold, in order to design more potent NQO1 inhibitors, which would exhibit highly potent antitumor activity.
Assuntos
Simulação de Acoplamento Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Relação Quantitativa Estrutura-Atividade , Quinonas/metabolismo , Sítios de Ligação , Desenho Assistido por Computador , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Análise dos Mínimos Quadrados , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Estrutura Terciária de Proteína , Quinonas/química , Eletricidade EstáticaRESUMO
To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP(+) than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.
Assuntos
Catálise , Coenzimas/química , Flavina-Adenina Dinucleotídeo/química , NADH NADPH Oxirredutases/química , Rhodobacter capsulatus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/metabolismo , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/química , Flavinas/metabolismo , Flavodoxina/química , Mutação , NADH NADPH Oxirredutases/metabolismo , NADP/química , Dobramento de Proteína , PrótonsRESUMO
Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 °C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in â¼5-10 °C the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , NADH Desidrogenase/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/química , Flavina-Adenina Dinucleotídeo/química , Cinética , NADH Desidrogenase/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
Thiol-covered nanostructured gold has been tested as a platform for the preparation of high-area phospholipid bilayer systems suitable for optical and electrochemical sensing. In situ and ex situ Raman spectroscopy and electrochemical measurements are made to study methylene blue (MB) and flavin-adenine dinucleotide (FAD) incorporation into dimyristoylphosphatidylcholine (DMPC) bilayers prepared by vesicle fusion on dithiothreitol (DTT)-covered nanostructured gold. Results show that lipophilic positively charged MB molecules are incorporated in the bilayer reaching the DTT-gold interface. On the other hand, the negatively charged FAD molecules are immobilized at the outer part of the phospholipid bilayer and cannot be electrochemically detected. Our results demonstrate that DTT-covered nanostructured gold provides a suitable high-area platform for phospholipid membranes that are able to separate and sense different kinds of molecules and biomolecules.
Assuntos
Dimiristoilfosfatidilcolina/química , Ditiotreitol/química , Flavina-Adenina Dinucleotídeo/química , Ouro/química , Bicamadas Lipídicas/química , Azul de Metileno/química , Eletroquímica , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Oxirredução , Análise Espectral Raman , Compostos de Sulfidrila/químicaRESUMO
Self-assembly of dithiothreitol (DTT) on Au(111) from solution deposition has been studied by X-ray photoelectron spectroscopy and electrochemical data. DTT molecules self-assemble on Au(111) in a lying-down configuration irrespective of the concentration and temperature. XPS and electrochemical data indicate a DTT surface coverage of theta approximately 0.16 with two S-head-Au covalent bonds per DTT molecule. The DTT monolayer turns the Au surface hydrophilic enough to allow the formation of fluid dimyristoylphosphatidylcholine (DMPC) bilayer domains by vesicle fusion as revealed by in situ atomic force imaging. Methylene blue (MB) and flavin adenine dinucleotide (FAD) have been used as probes to study molecule transport across the bilayer.
Assuntos
Ditiotreitol/química , Ouro/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Dimiristoilfosfatidilcolina/química , Ditiotreitol/farmacologia , Eletroquímica , Flavina-Adenina Dinucleotídeo/química , Azul de Metileno/química , Sondas Moleculares/química , Análise Espectral , Enxofre/química , Propriedades de SuperfícieRESUMO
We report a fully integrated core-shell nanoparticle system responsive to glucose. The system is comprised of self-assembled glucose oxidase and an osmium molecular wire on core-shell Au nanoparticles. Characterization of the functional nanoparticles by spectroscopy, quartz crystal microbalance and electrochemical techniques has shown that the catalytically active shell has a structure as designed and all components are active in the self-assembled multilayer shell. Furthermore, amperometric reagentless detection of glucose and contactless photonic biosensing by the Os(II) resonant Raman signal have been demonstrated. The enzymatic reduction of FAD by glucose and further reduction of the Raman silent Os(III) by FADH 2 yields a characteristic enzyme-substrate calibration curve in the millimolar range. Furthermore, coupling of electronic resonant Raman of the osmium complex with the SERS amplification by Au NPs plasmon resonance has been demonstrated which leads to an extra enhancement of the biosensor signal. We present a proof of concept extending the work done with planar surfaces to core-shell NPs as an advance in the design of glucose-responsive chemistry detected by SERS-like methods.