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1.
Biosci Biotechnol Biochem ; 84(4): 789-796, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31889476

RESUMO

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC50A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val17 was not responsible for the enzyme inactivation in the Ala17Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala17Val mutant. It is, therefore, reasonable to assume that Ala17 residue is critical for PAI functionality.Abbreviations: pai: propionibacterium acnes isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.


Assuntos
Substituição de Aminoácidos , Flavina-Adenina Dinucleotídeo/metabolismo , Isomerases/antagonistas & inibidores , Propionibacterium acnes/enzimologia , Western Blotting , Códon , Cristalografia por Raios X , Isomerases/química , Isomerases/genética , Isomerases/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Mutação com Perda de Função , Propionibacterium acnes/genética , Conformação Proteica
2.
Biochim Biophys Acta Bioenerg ; 1861(1): 148118, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734195

RESUMO

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH-. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.


Assuntos
Aldeído Oxirredutases/química , Bradyrhizobiaceae/enzimologia , Metaloproteínas/química , Complexos Multienzimáticos/química , Aldeído Oxirredutases/metabolismo , Catálise , Domínio Catalítico , Cobalto/química , Cobalto/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Metaloproteínas/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Complexos Multienzimáticos/metabolismo
3.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31614972

RESUMO

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.


Assuntos
Corynebacterium/enzimologia , Óxido Nítrico Sintase/química , Nucleotidiltransferases/química , Termodinâmica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico/genética , Corynebacterium/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Ligantes , Modelos Moleculares , Óxido Nítrico Sintase/genética , Nucleotidiltransferases/genética , Especificidade por Substrato
4.
Methods Appl Fluoresc ; 7(4): 044005, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31553966

RESUMO

Autofluorescence based fluorescence lifetime imaging microscopy (AF-FLIM) techniques have come a long way from early studies on cancer characterization and have now been widely employed in several cellular and animal studies covering a wide range of diseases. The majority of research in autofluorescence imaging (AFI) study metabolic fluxes in live biological samples. However, tissues from clinical or scientific studies are often chemically fixed for preservation and stabilization of tissue morphology. Fixation is particularly crucial for enzymatic, functional, or histopathology studies. Interpretations of metabolic imaging such as optical redox intensity imaging and AF-FLIM, have often been viewed as potentially unreliable in a fixed sample due to lack of studies in this field. In this study, we carefully evaluate the possibility of extracting microenvironment information in fixed tissues using reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) endogenous fluorescence. The ability to distinguish changes such as metabolism and pH using intrinsic fluorescence in fixed tissues has great pathological value. In this work, we show that the lifetime based metabolic contrast in a sample is preserved after chemical fixation. The fluorescence lifetime of a sample increases with an additive fixative like formaldehyde; however, the fixed tissues retain metabolic signatures even after fixation. This study presents an opportunity to successfully image archived unstained histopathology tissues, and generate useful AF-FLIM signatures. We demonstrate the capability to draw metabolic interpretations in fixed tissues even after long periods of storage.


Assuntos
Microscopia de Fluorescência , NADP/metabolismo , NAD/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Flavina-Adenina Dinucleotídeo/metabolismo , Formaldeído , Humanos , Camundongos , Mitocôndrias/metabolismo , Inclusão em Parafina , Fatores de Tempo , Fixação de Tecidos
5.
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
6.
Microbiology ; 165(10): 1095-1106, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31339487

RESUMO

Dodecins are small flavin-binding proteins that are widespread amongst haloarchaeal and bacterial species. Haloarchaeal dodecins predominantly bind riboflavin, while bacterial dodecins have been reported to bind riboflavin-5'-phosphate, also called flavin mononucleotide (FMN), and the FMN derivative, flavin adenine dinucleotide (FAD). Dodecins form dodecameric complexes and represent buffer systems for cytoplasmic flavins. In this study, dodecins of the bacteria Streptomyces davaonensis (SdDod) and Streptomyces coelicolor (ScDod) were investigated. Both dodecins showed an unprecedented low affinity for riboflavin, FMN and FAD when compared to other bacterial dodecins. Significant binding of FMN and FAD occurred at relatively low temperatures and under acidic conditions. X-ray diffraction analyses of SdDod and ScDod revealed that the structures of both Streptomyces dodecins are highly similar, which explains their similar binding properties for FMN and FAD. In contrast, SdDod and ScDod showed very different properties with regard to the stability of their dodecameric complexes. Site-directed mutagenesis experiments revealed that a specific salt bridge (D10-K62) is responsible for this difference in stability.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Streptomyces coelicolor/química , Streptomyces/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Multimerização Proteica , Estabilidade Proteica , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Especificidade da Espécie , Streptomyces/genética , Streptomyces coelicolor/genética , Temperatura
7.
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
8.
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
9.
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
10.
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 , Imageamento Tridimensional , Rim/metabolismo , Nefropatias/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Lesões por Radiação/metabolismo , Ratos
11.
Biochemistry ; 58(18): 2362-2372, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30964996

RESUMO

There is an increasing realization that structure-based drug design may show improved success by understanding the ensemble of conformations accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson's disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear, and routes to drugging this target would be valuable. Evidence of a radical in either the transition state or the resting state of MAO-B is present throughout the literature and is suggested to be a flavin semiquinone, a tyrosyl radical, or both. Here we see evidence of a resting-state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. On the basis of enzyme kinetic studies, enzyme variants, and molecular dynamics simulations, we find evidence for the importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity and protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these targets.


Assuntos
Membrana Celular/química , Flavina-Adenina Dinucleotídeo/análogos & derivados , Simulação de Dinâmica Molecular , Monoaminoxidase/química , Sítios de Ligação , Domínio Catalítico , Membrana Celular/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Cinética , Monoaminoxidase/metabolismo , Oxirredução , Ligação Proteica
12.
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
13.
Nat Commun ; 10(1): 1255, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890712

RESUMO

Controlled halogenation of chemically versatile substrates is difficult to achieve. Here we describe a unique flavin-dependent halogenase, PltM, which is capable of utilizing a wide range of halides for installation on a diverse array of phenolic compounds, including FDA-approved drugs and natural products, such as terbutaline, fenoterol, resveratrol, and catechin. Crystal structures of PltM in complex with phloroglucinol and FAD in different states yield insight into substrate recognition and the FAD recycling mechanism of this halogenase.


Assuntos
Proteínas de Bactérias/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Halogenação , Modelos Moleculares , Mutagênese , Oxirredutases/genética , Floroglucinol/química , Floroglucinol/metabolismo , Especificidade por Substrato
14.
Appl Biochem Biotechnol ; 188(4): 897-913, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30729393

RESUMO

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-D-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47 kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71 U/mg at optimum pH and temperature of 6.0 and 35 °C. The Km and Vmax values of calcium D-gluconate were 0.631 mM and 0.734 mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement.


Assuntos
Gluconatos/metabolismo , Oxirredutases/metabolismo , Pseudomonas/metabolismo , Citocromos c/metabolismo , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular
15.
Biochemistry ; 58(13): 1751-1763, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30810301

RESUMO

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.


Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/metabolismo , Oxigenases de Função Mista/metabolismo , Niacina/metabolismo , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/enzimologia , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Hidroxilação , Cinética , Ácidos Nicotínicos/metabolismo , Parabenos/metabolismo , Piridinas/metabolismo , Especificidade por Substrato
16.
Talanta ; 197: 105-112, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30771910

RESUMO

The simultaneous quantitative analysis of intracellular metabolic coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) is of interest because they participate in many electron-transfer reactions of metabolism. But, the simultaneous quantitative analysis of FAD and FMN is hard to be achieved by traditional analytical methods. This paper proposes a novel strategy of intrinsic fluorescence coupled with four-way calibration method for simultaneous quantitative analysis of intracellular metabolic coenzymes FAD and FMN. Through mathematical separation, this proposed analytical method efficiently achieved the simultaneous quantitative analysis of metabolic coenzymes FAD and FMN in the cell, despite the fact that uncalibrated spectral interferents coexist in the system. The predicted concentrations of FAD and FMN in the cell are 217.0 ±â€¯6.9 and 155.0 ±â€¯1.7 pmol/106 cells respectively, which were validated by the approved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. This analytical method with second-order advantage simply requires the cell solution to be diluted by a buffer, it could introduce an interesting analytical strategy for multianalyte direct quantitative analysis in complex biological systems. In addition, we explore the third-order advantage of four-way calibration by a comparative study based on this real fluorescence data. The comparisons indicate that a four-way calibration method can provide higher sensitivity and more resolving power than a three-way calibration method.


Assuntos
Mononucleotídeo de Flavina/análise , Flavina-Adenina Dinucleotídeo/análise , Fluorescência , Calibragem , Cromatografia Líquida , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Células HeLa , Humanos , Espectrometria de Massas em Tandem
17.
Life Sci Alliance ; 2(1)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30659069

RESUMO

d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.


Assuntos
Senescência Celular/fisiologia , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aminoácidos/metabolismo , Arginina/metabolismo , Senescência Celular/efeitos dos fármacos , Coenzimas/metabolismo , D-Aminoácido Oxidase/antagonistas & inibidores , Dano ao DNA/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Oxirredução , Interferência de RNA , Riboflavina/farmacologia , Serina/metabolismo , Transfecção
18.
Int J Biol Macromol ; 126: 1223-1233, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615965

RESUMO

Ion binding to biomacromolecules can modulate their activity and stability in vivo. It is of particular interest to understand the structural and energetic basis of anion binding to functional sites of biomacromolecules. In this work, binding of anions to the FAD binding pocket of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with cancer due to a common polymorphism causing a P187S amino acid substitution, was investigated. It is known that NQO1 stability in vivo is strongly modulated by binding of its flavin cofactor. Herein, binding and protein stability analyses were carried out to show that anion binding to the apo-state of NQO1 P187S inhibits FAD binding with increasing strength following the chaotropic behavior of anions. These inhibitory effects were significant for some anions even at low millimolar concentrations. Additional pH dependent analyses suggested that protonation of histidine residues in the FAD binding pocket was not critical for anion or flavin binding. Overall, this detailed biophysical analysis helps to understanding how anions modulate NQO1 functionality in vitro, thus allowing hypothesize that NQO1 stability in vivo could be modulated by differential anion binding and subsequent inhibition of FAD binding.


Assuntos
Flavinas/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ânions , Flavina-Adenina Dinucleotídeo/metabolismo , Histidina/metabolismo , Humanos , Cinética , Prótons , Temperatura , Água
19.
Org Biomol Chem ; 17(6): 1506-1518, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30681110

RESUMO

The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.


Assuntos
Antimaláricos/metabolismo , Fosfomicina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Biocatálise , Flavina-Adenina Dinucleotídeo/metabolismo , Fosfomicina/biossíntese , Cinética , Oxigenases de Função Mista/química , Modelos Moleculares , NADP/metabolismo , Domínios Proteicos
20.
Fertil Steril ; 111(2): 212-218, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30691624

RESUMO

Current strategies for embryo assessment in the assisted reproductive technology laboratories rely primarily on morphologic parameters that have limited accuracy for determining embryo viability. Even with the addition of invasive diagnostic interventions such as preimplantation genetic testing for aneuploidy alone or in combination with mitochondrial DNA copy number assessment, at least one third of embryos fail to implant. Therefore, at a time when the clinical benefits of single ET are widely accepted, improving viability assessment of embryos is ever more important. Building on the previous work demonstrating the importance of metabolic state in oocytes and embryos, metabolic imaging via fluorescence lifetime imaging microscopy offers new and potentially useful diagnostic method by detecting natural fluorescence of FAD and NADH, the two electron transporters that play a central role in oxidative phosphorylation. Recent studies demonstrate that fluorescence lifetime imaging microscopy can detect oocyte and embryo metabolic function and dysfunction in a multitude of experimental models and provide encouraging evidence for use in scientific investigation and possibly for clinical application.


Assuntos
Blastocisto/metabolismo , Metabolismo Energético , Fertilização In Vitro , Infertilidade/terapia , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Oócitos/metabolismo , Biomarcadores/metabolismo , Blastocisto/patologia , Sobrevivência Celular , Feminino , Fertilidade , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Infertilidade/metabolismo , Infertilidade/patologia , Infertilidade/fisiopatologia , Masculino , Mitocôndrias/patologia , NAD/metabolismo , Oócitos/patologia , Valor Preditivo dos Testes , Gravidez , Resultado do Tratamento
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