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1.
Methods Mol Biol ; 2280: 275-295, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751442

RESUMO

The aim of this short review chapter is to provide a brief summary of the relevance of riboflavin (Rf or vitamin B2) and its derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for human neuromuscular bioenergetics.Therefore, as a completion of this book we would like to summarize what kind of human pathologies could derive from genetic disturbances of Rf transport, flavin cofactor synthesis and delivery to nascent apoflavoproteins, as well as by alteration of vitamin recycling during protein turnover.


Assuntos
Músculo Esquelético/metabolismo , Neurônios/metabolismo , Riboflavina/metabolismo , Metabolismo Energético , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Predisposição Genética para Doença , Humanos , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo
2.
Biochimie ; 182: 217-227, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33516756

RESUMO

Flavin adenine dinucleotide synthetase (FADS), a bifunctional prokaryotic enzyme, is involved in the synthesis of two vital cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here, we investigated the biochemical characteristics of FADS from Staphylococcus aureus (Sa), a pathogenic bacteria causing food-borne diseases. The SaFADS possesses riboflavin kinase (RFK) and FMN adenylyltransferase (FMNAT) activities that transforms riboflavin to FMN and FMN to FAD, respectively. The FMNAT domain also exhibits reversible FAD pyrophosphorylase activity (FADpp). Further, we show that the FMNAT and FADpp activities are dependent on the reducing environment. Mutations of the conserved K289 and F290 residues present on the RFK domain affect the kinetic parameters of both the RFK and FMNAT domains. Additionally, the molecular dynamics analysis of apo and riboflavin: ATP: Mg2+ ternary complex of SaFADS shows that F290 is involved in stabilizing the active site geometry to hold the enzyme-substrate complex. In addition, the deletion of the αh2 helix that acts as a connecting linker between the FMNAT and RFK domains showed substantial loss of their activities. The helix deletion could have affected the flap motion of L2c, L4c, ß4n and L3n present in the close proximity resulting in the distortion of the active site geometry. In conclusion, our study has characterized the RFK and FMNAT activities of SaFADS and shown the importance of conserved K289 and F290 in RFK activity. As FADSs are potential drug targets, understanding their mechanism of action might help in discovering species-specific antibacterial drugs.


Assuntos
Proteínas de Bactérias/química , Nucleotidiltransferases/química , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Riboflavina/química , Riboflavina/metabolismo , Staphylococcus aureus/genética , Especificidade por Substrato
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 246: 118997, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33032115

RESUMO

The present study describes a comprehensive investigation of the spectroscopic characteristics, stability and in vitro antioxidant and cytotoxic properties of the Flavin MonoNucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) in Dextran70 (Dx70) and Dx70/phospatidylcholine (PC) biomimetic systems by means of the UV-Vis absorption, fluorescence spectroscopy, chemiluminescence and Neutral Red assay. The affinity of FMN, FAD and the precursor riboflavin (RF) to an unsaturated phospholipid bilayer model as well as the location of the probes within the lipid bilayer were assessed from united-atom molecular dynamics simulations carried out on an unsaturated phospholipid bilayer model system, and the theoretical and experimental characterization of the two probes within biomembranes was complemented with the light microscopy survey of the cell morphology of L929 fibroblast cells cultivated in the presence of various dosage of FAD/FMN. In lipid bilayers, FMN/FAD resulted in a noticeable improvement of the antioxidant activity (the scavenging of reactive oxygen species up to 40%) and a significant effect on cellular viability in the L929 fibroblast cells. The results are important in the oxidative stress process concerning the redox reactions of flavins in humans as well as in further studies on different systems belonging to the category of flavoenzymes/flavoproteins, required for cellular respiration.


Assuntos
Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo , Biomimética , Mononucleotídeo de Flavina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Riboflavina
4.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32737127

RESUMO

Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a "butterfly bend" conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products.IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.


Assuntos
Proteínas de Bactérias/química , Redes e Vias Metabólicas , Nicotina/metabolismo , Pseudomonas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mononucleotídeo de Flavina/metabolismo , Pseudomonas/enzimologia , Piridinas/metabolismo , Pirrolidinas/metabolismo , Alinhamento de Sequência
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
J Mol Biol ; 431(22): 4523-4526, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31473159

RESUMO

Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional unit hosts an α-subunit (αGltS) and a ß-subunit (ßGltS) that assemble in different αß oligomers in solution. Here, we present the cryo-electron microscopy structures of Azospirillum brasilense GltS in four different oligomeric states (α4ß3, α4ß4, α6ß4 and α6ß6, in the 3.5- to 4.1-Å resolution range). Our study provides a comprehensive GltS model that details the inter-protomeric assemblies and allows unequivocal location of the FAD cofactor and of two electron transfer [4Fe-4S]+1,+2 clusters within ßGltS.


Assuntos
Azospirillum brasilense/enzimologia , Microscopia Crioeletrônica/métodos , Glutamato Sintase/metabolismo , Glutamato Sintase/ultraestrutura , Catálise , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/ultraestrutura
13.
J Biol Inorg Chem ; 24(6): 849-861, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31410573

RESUMO

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.


Assuntos
Bacillus anthracis/enzimologia , Bacillus anthracis/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Metaloproteases/metabolismo , Cristalografia por Raios X , FMN Redutase/química , FMN Redutase/genética , FMN Redutase/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Mononucleotídeo de Flavina/metabolismo , Metaloproteases/química , Metaloproteases/genética , Ribonucleotídeo Redutases
14.
Mol Biochem Parasitol ; 233: 111202, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31381947

RESUMO

Chorismate synthase (Cs) is the last enzyme of the main trunk of shikimate pathway and catalyzes formation of chorismate, a major aromatic metabolite precursor. We have previously reported that Cs is highly conserved across different Plasmodium sp. Here we report that Cs from malaria parasites are bifunctional enzymes through expression and functional studies of two recombinant proteins rPfCs (Cs from P. falciparum) and rPvCs (Cs from P. vivax). We confirm bifunctional activity of both rPfCs and rPvCs based on their ability to catalyze formation of chorismate under aerobic conditions as well as their ability to catalyze generation of reduced flavin mononucleotide (FMN) as assessed through diaphorase assay.


Assuntos
Fósforo-Oxigênio Liases , Plasmodium falciparum/enzimologia , Plasmodium vivax/enzimologia , Mononucleotídeo de Flavina/metabolismo , Malária/parasitologia , Fósforo-Oxigênio Liases/metabolismo , Proteínas Recombinantes/metabolismo , Ácido Chiquímico/metabolismo
15.
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
16.
J Biol Inorg Chem ; 24(6): 889-898, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31463592

RESUMO

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.


Assuntos
Cupriavidus necator/enzimologia , Cupriavidus necator/metabolismo , Formiato Desidrogenases/metabolismo , Catálise , Coenzimas/metabolismo , Cupriavidus necator/genética , Mononucleotídeo de Flavina/metabolismo , Formiato Desidrogenases/química , Formiato Desidrogenases/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Metaloproteínas/metabolismo , Oxirredução , Pteridinas/metabolismo
17.
Arch Biochem Biophys ; 673: 108080, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31445894

RESUMO

The electron configuration of flavin cofactors, FMN and FAD, is a critical factor governing the reactivity of NADPH-cytochrome P450 reductase (CPR). The current view of electron transfer by the mammalian CPR, based on equilibrium redox potentials of the flavin cofactors, is that the two electron-reduced FMN hydroquinone (FMNH2), rather than one electron-reduced FMN semiquinone, serves as electron donor to the terminal protein acceptors. However, kinetic and thermodynamic studies on the CPR species originated from different organisms have shown that redox potentials measured at distinct electron transfer steps differ from redox potentials determined by equilibrium titration. Collectively, previous observations suggest that the short-lived transient semiquinone species may carry electrons in diflavin reductases. In this work, we have investigated spectroscopic properties of the CPR-bound FAD and FMN reduced at 77 K by radiolytically-generated thermalized electrons. Using UV-vis spectroscopy, we demonstrated that upon cryo-reduction of oxidized yeast CPR (yCPR) containing an equimolar ratio of both FAD and FMN, or FAD alone, neutral semiquinones were trapped at 77 K. During annealing at the elevated temperatures, unstable short-lived neutral semiquinones relaxed to spectroscopically distinct air-stable neutral semiquinones. This transition was independent of pH within the 6.0-10.7 range. Our data on yeast CPR are in line with the previous observations of others that the flavin short-lived transient semiquinone intermediates may have a role in the electron transfer by CPR at physiological conditions.


Assuntos
Flavina-Adenina Dinucleotídeo/análogos & derivados , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Temperatura , Leveduras/enzimologia , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose Oxidase/metabolismo , Cinética , Oxirredução
18.
FEMS Microbiol Lett ; 366(12)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31291453

RESUMO

Fluorescent signals associated with Complex I (NADH:ubiquinone oxidoreductase type I) upon its reduction by NADH without added acceptors and upon NADH:ubiquinone oxidoreduction were studied. Two Complex I-associated redox-dependent signals were observed: with maximum emission at 400 nm (λex = 320 nm) and 526 nm (λex = 450 nm). The 400 nm signal derived from ubiquinol accumulated in Complex I/DDM (n-dodecyl ß-D-maltopyranoside) micelles. The 526 nm redox signal unexpectedly derives mainly from FMN (flavin mononucleotide), whose fluorescence in oxidized protein is fully quenched, but arises transiently upon reduction of Complex I by NADH. The paradoxical flare-up of FMN fluorescence is discussed in terms of conformational changes in the catalytic site upon NADH binding. The difficulties in revealing semiquinone fluorescent signal are considered.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Domínio Catalítico , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Concentração de Íons de Hidrogênio , NAD/química , NAD/metabolismo , Oxirredução
19.
Biochem Biophys Res Commun ; 516(4): 1211-1215, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31300198

RESUMO

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.


Assuntos
Dinitrocresóis/metabolismo , Mitocôndrias Hepáticas/metabolismo , Riboflavina/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Nucleotídeos de Guanina/metabolismo , Homeostase , Hidrólise , Íons , Ferro/metabolismo , Luminescência , Niacinamida/metabolismo , Ratos , Ratos Wistar , Superóxidos/metabolismo
20.
J Biol Chem ; 294(37): 13800-13810, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31350338

RESUMO

The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pK ES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. We propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.


Assuntos
Flavinas/metabolismo , Transferases/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Sequência Conservada , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/genética , Histidina/metabolismo , Cinética , Oxirredução , Especificidade por Substrato/genética , Transferases/genética , Vibrio cholerae/genética
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