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1.
Nat Commun ; 11(1): 867, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054832

RESUMO

Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-ß peptides (Aß) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aß42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aß42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aß42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Mononucleotídeo de Flavina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Genes Sintéticos , Genoma Fúngico , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Genéticos , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Dobramento de Proteína , Proteólise , RNA-Seq , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
2.
Phys Chem Chem Phys ; 22(12): 6538-6552, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-31994556

RESUMO

Flavin mononucleotide (FMN) belongs to the large family of flavins, ubiquitous yellow-coloured biological chromophores that contain an isoalloxazine ring system. As a cofactor in flavoproteins, it is found in various enzymes and photosensory receptors, like those featuring the light-oxygen-voltage (LOV) domain. The photocycle of FMN is triggered by blue light and proceeds via a cascade of intermediate states. In this work, we have studied isolated FMN in an aqueous solution in order to elucidate the intrinsic electronic and vibrational changes of the chromophore upon excitation. The ultrafast transitions of excited FMN were monitored through the joint use of femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy encompassing a time window between 0 ps and 6 ns with 50 fs time resolution. Global analysis of the obtained transient visible absorption and transient Raman spectra in combination with extensive quantum chemistry calculations identified unambiguously the singlet and triplet FMN populations and addressed solvent dynamics effects. The good agreement between the experimental and theoretical spectra facilitated the assignment of electronic transitions and vibrations. Our results represent the first steps towards more complex experiments aimed at tracking structural changes of FMN embedded in light-inducible proteins upon photoexcitation.


Assuntos
Mononucleotídeo de Flavina/química , Processos Fotoquímicos , Análise Espectral Raman , Simulação por Computador , Mononucleotídeo de Flavina/metabolismo
3.
Appl Microbiol Biotechnol ; 104(5): 2051-2066, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31930452

RESUMO

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.


Assuntos
Extremófilos/enzimologia , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Alcenos/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cianobactérias/enzimologia , Cianobactérias/genética , Cianobactérias/metabolismo , Bases de Dados Genéticas , Estabilidade Enzimática , Extremófilos/genética , Extremófilos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , NADP/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/isolamento & purificação , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodófitas/enzimologia , Rodófitas/genética , Especificidade por Substrato
4.
Photochem Photobiol Sci ; 18(11): 2657-2660, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624823

RESUMO

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.


Assuntos
Proteínas de Bactérias/metabolismo , Methylobacterium/metabolismo , Oxigênio Singlete/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Polarização de Fluorescência , Mutagênese Sítio-Dirigida , Oxigênio/química , Ligação Proteica , Teoria Quântica , Alinhamento de Sequência , Ureia/química
5.
J Agric Food Chem ; 67(43): 12037-12043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31581772

RESUMO

Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV (Q = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mononucleotídeo de Flavina/metabolismo , Fluorescência , Luz , Domínios Proteicos , Estabilidade Proteica
6.
Enzyme Microb Technol ; 131: 109433, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615666

RESUMO

Azoreductase from Chromobacterium violaceum was characterized biophysically using experimental and computational tools. The in-silico docking and cross-linking experiments using glutaraldehyde suggest dimeric nature of the enzyme. The enzyme structure was modelled and also studied using circular dichroism (CD) spectroscopy which suggests 40% α- helix, 30% ß- sheet and 30% random coils. In the modelled structure of the azoreductase, the cofactor flavin mononucleotide (FMN) binding energy was -3.8 kJ/mol. The binding of FMN affects the azoreductase-cofactor complex stability. The stability-folding studies indicate that the cofactor, FMN is required for folding, stability and activity. Overall, the data provides interesting insight into stability and biophysical parameters of the azoreductase protein.


Assuntos
Chromobacterium/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Dobramento de Proteína , Dicroísmo Circular , Coenzimas/metabolismo , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Estabilidade Proteica
7.
J Biotechnol ; 305: 18-22, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31472166

RESUMO

C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.


Assuntos
Álcool Desidrogenase/metabolismo , Clostridium tyrobutyricum/crescimento & desenvolvimento , Mononucleotídeo de Flavina/metabolismo , 2-Propanol/metabolismo , Álcool Desidrogenase/genética , Biocombustíveis , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/crescimento & desenvolvimento , Clostridium acetobutylicum/metabolismo , Clostridium tyrobutyricum/genética , Clostridium tyrobutyricum/metabolismo , Fluorescência , Genes Reporter , Engenharia Genética , Regiões Promotoras Genéticas
8.
Acta Crystallogr D Struct Biol ; 75(Pt 8): 733-742, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373572

RESUMO

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.


Assuntos
Oxirredutases do Álcool/química , Mononucleotídeo de Flavina/metabolismo , Ácidos Mandélicos/metabolismo , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular/métodos , Cristalografia por Raios X/métodos , Descarboxilação , Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Oxirredução , Ligação Proteica , Especificidade por Substrato
9.
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
10.
Methods Enzymol ; 620: 145-166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072485

RESUMO

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed.


Assuntos
Ensaios Enzimáticos/métodos , Marcação por Isótopo/métodos , Oxirredutases/química , Dicroísmo Circular , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Cinética , Isótopos de Nitrogênio/química , Oxirredutases/genética , Oxirredutases/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Int J Biol Macromol ; 132: 254-264, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928375

RESUMO

Dihydrouridination is one of the abundant modifications in tRNA editing. The presence of dihydrouridine is attributed to tRNA stability desired for the efficient gene translation process. The conversion of uridine to dihydrouridine is catalyzed by flavine containing enzyme called dihydrouridine synthase (Dus). We report first ever information about DusA enzyme from Pseudomonas aeruginosa in form of structural and functional studies. The gene coding for DusA from P. aeruginosa (PADusA) was cloned, expressed and purified, using recombinant DNA technology methods. Thermal and chemical stability of PADusA was determined with respect to temperature and urea-induced equilibrium unfolding experiments, with monitoring the change of ellipticity at 200-260 nm by Circular Dichroism (CD) spectroscopy. Unfolding studies revealed that PADusA has acquired a stable tertiary structure fold with a Tm value of 46.2 °C and Cm of 2.7 M for urea. The enzyme contains 43% α-helices and 16% ß-strands. The three dimensional structure of PADusA was modeled using insilico methods. In order to understand the mechanism of substrate recognition and catalysis, tRNA and puromycin were docked on PADusA structure and their binding was analyzed. The structural features suggested that PADusA may also form a novel target for structure based drug design of antimicrobial agents.


Assuntos
Oxirredutases/química , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Mononucleotídeo de Flavina/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Oxirredutases/metabolismo , Domínios Proteicos , Dobramento de Proteína , Puromicina/metabolismo , RNA de Transferência/metabolismo , Termodinâmica
12.
J Biochem ; 166(1): 67-75, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715389

RESUMO

Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification.


Assuntos
Aspergillus oryzae/enzimologia , Oxirredutases/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Oxirredutases/química
13.
Bioelectrochemistry ; 127: 94-103, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30771661

RESUMO

Biofilm-coated electrodes and outer cell membrane-mimicked electrodes were examined to verify an extracellular electron transfer mechanism using Marcus theory for a donor-acceptor electron transfer. Redox couple-bound membrane electrodes were prepared by impregnating redox coenzymes into Nafion films on carbon cloth electrodes. The electron transfer was believed to occur sequentially from acetate to nicotinamide adenine dinucleotide (NAD), c-type cytochrome, flavin mononucleotide (FMN) (or riboflavin (RBF)) and the anode substrate. Excellent polarisation and power density characteristics were contributed by the modification of the cathode with a high-surface-area ordered mesoporous carbon or a hollow core-mesoporous shell carbon. The maximum power density of the microbial fuel cell (MFC) could be improved by a factor of two mainly due to the accelerated electron consumption by modifying the cathode surfaces within three-dimensionally interconnected mesoporous carbon particles, and the anode was coated with a mixed culture of anaerobic bacteria.


Assuntos
Fontes de Energia Bioelétrica/microbiologia , Acetatos/metabolismo , Biofilmes/crescimento & desenvolvimento , Carbono/química , Clostridium/enzimologia , Clostridium/fisiologia , Citocromos c/metabolismo , Eletricidade , Eletrodos , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , NAD/metabolismo , Oxirredução , Porosidade , Proteobactérias/enzimologia , Proteobactérias/fisiologia
14.
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
15.
RNA ; 25(1): 23-34, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30287481

RESUMO

Many bacteria use flavin mononucleotide (FMN) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor. Representatives of one of the two variant subtypes were found to bind the FMN precursor riboflavin and the FMN degradation products lumiflavin and lumichrome. Although the biologically relevant ligand sensed by these variant FMN riboswitches remains uncertain, our findings suggest that many strains of C. difficile might use rare riboswitches to sense flavin degradation products and activate transporters for their detoxification.


Assuntos
Clostridium difficile/genética , Clostridium difficile/metabolismo , Mononucleotídeo de Flavina/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Riboswitch/genética , Sequência de Bases , Clostridium difficile/classificação , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Variação Genética , Ligantes , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Fotólise , Especificidade da Espécie
16.
Z Naturforsch C J Biosci ; 74(3-4): 101-104, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30379645

RESUMO

There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.


Assuntos
Proteínas de Bactérias/química , FMN Redutase/química , Formiato Desidrogenases/química , Proteínas Fúngicas/química , Peróxido de Hidrogênio/síntese química , Oxigenases de Função Mista/química , Agrocybe/química , Agrocybe/enzimologia , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Biocatálise , Candida/química , Candida/enzimologia , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Coenzimas/química , Coenzimas/metabolismo , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Formiato Desidrogenases/metabolismo , Formiatos/química , Formiatos/metabolismo , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/metabolismo , NAD/química , NAD/metabolismo , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Estereoisomerismo
17.
Nat Commun ; 9(1): 4867, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451826

RESUMO

Osm1 and Frd1 are soluble fumarate reductases from yeast that are critical for allowing survival under anaerobic conditions. Although they maintain redox balance during anaerobiosis, the underlying mechanism is not understood. Here, we report the crystal structure of a eukaryotic soluble fumarate reductase, which is unique among soluble fumarate reductases as it lacks a heme domain. Structural and enzymatic analyses indicate that Osm1 has a specific binding pocket for flavin molecules, including FAD, FMN, and riboflavin, catalyzing their oxidation while reducing fumarate to succinate. Moreover, ER-resident Osm1 can transfer electrons from the Ero1 FAD cofactor to fumarate either by free FAD or by a direct interaction, allowing de novo disulfide bond formation in the absence of oxygen. We conclude that soluble eukaryotic fumarate reductases can maintain an oxidizing environment under anaerobic conditions, either by oxidizing cellular flavin cofactors or by a direct interaction with flavoenzymes such as Ero1.


Assuntos
Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Glicoproteínas/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Riboflavina/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Succinato Desidrogenase/química , Anaerobiose/genética , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Shewanella/enzimologia , Shewanella/genética , Especificidade por Substrato , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Triazinas/química , Triazinas/metabolismo
18.
J Nutr Biochem ; 62: 123-133, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30290331

RESUMO

Involvement of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding retinal pigment epithelium (RPE). Optimizations led to detection efficiency of 0.1 pmol for FAD and FMN while 0.01 pmol for riboflavin. Interestingly, levels of FAD and FMN in the RPE were found to be 1.7- and 12.5-fold higher than their levels in the retina, respectively. Both FAD and FMN levels in the RPE and retina gradually decline with age and preceded the age-dependent drop in the functional competence of the retina as measured by electroretinography. Further, quantifications of retinal levels of FAD and FMN in different mouse models of retinal degeneration revealed differential metabolic requirements of these two factors in relation to the rate and degree of photoreceptor degeneration. We also found twofold reductions in retinal levels of FAD and FMN in two mouse models of diabetic retinopathy. Altogether, our results suggest that retinal levels of FAD and FMN can be used as potential markers to determine state of health of the retina in general and more specifically the photoreceptors.


Assuntos
Dinitrocresóis/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Envelhecimento/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Jejum , Mononucleotídeo de Flavina/análise , Flavina-Adenina Dinucleotídeo/análise , Homeostase , Luz , Camundongos Endogâmicos C57BL , Epitélio Pigmentado da Retina/metabolismo
19.
Biophys J ; 115(6): 988-995, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30177440

RESUMO

Flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) are essential flavoprotein cofactors. A riboflavin kinase (RFK) activity catalyzes riboflavin phosphorylation to FMN, which can then be transformed into FAD by an FMN:adenylyltransferase (FMNAT) activity. Two enzymes are responsible for each one of these activities in eukaryotes, whereas prokaryotes have a single bifunctional enzyme, FAD synthase (FADS). FADS folds in two independent modules: the C-terminal with RFK activity and the N-terminal with FMNAT activity. Differences in structure and chemistry for the FMNAT catalysis among prokaryotic and eukaryotic enzymes pointed to the FMNAT activity of prokaryotic FADS as a potential antimicrobial target, making the structural model of the bacterial FMNAT module in complex with substrates relevant to understand the FADS catalytic mechanism and to the discovery of antimicrobial drugs. However, such a crystallographic complex remains elusive. Here, we have used molecular docking and molecular dynamics simulations to generate energetically stable interactions of the FMNAT module of FADS from Corynebacterium ammoniagenes with ATP/Mg2+ and FMN in both the monomeric and dimer-of-trimers assemblies reported for this protein. For the monomer, we have identified the residues that accommodate the reactive phosphates in a conformation compatible with catalysis. Interestingly, for the dimer-of-trimers conformation, we have found that the RFK module negatively influences FMN binding at the interacting FMNAT module. These results agree with calorimetric data of purified samples containing nearly 100% monomer or nearly 100% dimer-of-trimers, indicating that FMN binds to the monomer but not to the dimer-of-trimers. Such observations support regulation of flavin homeostasis by quaternary C. ammoniagenes FADS assemblies.


Assuntos
Biocatálise , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Coenzimas/metabolismo , Corynebacterium/enzimologia , Mononucleotídeo de Flavina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína
20.
ACS Chem Biol ; 13(9): 2728-2738, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30152678

RESUMO

Tautomycetin (TTN) is a polyketide natural product featuring a terminal alkene. Functional characterization of the genes within the ttn gene cluster from Streptomyces griseochromogenes established the biosynthesis of the TTN polyketide backbone, its dialkylmaleic anhydride moiety, the coupling of the two moieties to form the nascent intermediate TTN F-1, and the tailoring steps converting TTN F-1 to TTN. Here, we report biochemical and structural characterization of TtnD, a prenylated FMN (prFMN)-dependent decarboxylase belonging to the UbiD family that catalyzes the penultimate step of TTN biosynthesis. TtnD catalyzes decarboxylation of TTN D-1 to TTN I-1, utilizing prFMN as a cofactor generated by the TtnC flavin prenyltransferase; both TtnD and TtnC are encoded within the ttn biosynthetic gene cluster. TtnD exhibits substrate promiscuity but accepts only TTN D-1 congeners that feature an α,ß-unsaturated acid, supporting the [3+2] cycloaddition mechanism during catalysis that requires the double bond of an α,ß-unsaturated acid substrate. TtnD shares a similar overall structure with other members of the UbiD family but forms a homotetramer in solution. Each protomer is composed of three domains with the active site located between the middle and C-terminal domains; R169-E272-E277, constituting the catalytic triad, and E228, involved in Mn(II)-mediated binding of prFMN, were confirmed by site-directed mutagenesis. TtnD represents the first example of a prFMN-dependent decarboxylase involved in polyketide biosynthesis, expanding the substrate scope of the UbiD family of decarboxylases beyond simple aromatic and cinnamic acids. TtnD and its homologues are widespread in nature and could be exploited as biocatalysts for organic synthesis.


Assuntos
Vias Biossintéticas , Carboxiliases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Furanos/metabolismo , Streptomyces/enzimologia , Carboxiliases/química , Cristalografia por Raios X , Lipídeos , Modelos Moleculares , Conformação Proteica , Prenilação de Proteína , Streptomyces/química , Streptomyces/metabolismo , Especificidade por Substrato
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