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1.
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
2.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140303, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678192

RESUMO

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.


Assuntos
Mononucleotídeo de Flavina/química , NADPH Desidrogenase/química , Bacillus subtilis/enzimologia , Catálise , Cicloexanonas/química , Escherichia coli/genética , Mononucleotídeo de Flavina/efeitos da radiação , NADPH Desidrogenase/genética , NADPH Desidrogenase/efeitos da radiação , Oxirredução , Fotoquímica , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação
3.
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
4.
Acta Crystallogr D Struct Biol ; 75(Pt 10): 918-929, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31588923

RESUMO

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.


Assuntos
Oxirredutases do Álcool/química , Mononucleotídeo de Flavina/química , Actinobacteria/enzimologia , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Cinética , Mutação , Oxirredução , Especificidade por Substrato
5.
Methods Enzymol ; 620: 469-488, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072498

RESUMO

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.


Assuntos
Carboxiliases/metabolismo , Ensaios Enzimáticos/métodos , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/biossíntese , Aspergillus niger , Carboxiliases/isolamento & purificação , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/isolamento & purificação , Modelos Moleculares , Prenilação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Methods Enzymol ; 620: 489-508, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072499

RESUMO

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMNreduced species to the active prFMNiminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.


Assuntos
Carboxiliases/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Descarboxilação , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Oxirredução , Prenilação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
7.
Int J Pharm ; 564: 472-484, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30991131

RESUMO

Formulations for the controlled release of substances in the human terminal ileum and colon are essential to target the gut microbiome and its interactions with the intestinal mucosa. In contrast to pharmaceutical enteric coatings, reliable food-grade alternatives are still scarce. Shellac coatings have been used for various active ingredients, but their stability is affected by the physicochemical properties of the encapsulated substances. It is well known, that shellac release can be modulated by an acidic subcoating. Here, we hypothesized that a triple shellac coating with an adjusted intermediate coating (acidic or alkaline) can be effectively used to counteract the differences in pH value of various encapsulated substances, allowing a precise targeting of the desired release pH value. First, the system was tested with riboflavin 5'-monophosphate sodium salt dihydrate (RMSD) as a characteristic model substance. Secondly, it was transferred to nicotinic acid (NA) and nicotinamide (NAM) as bioactive compounds with different physio-chemical properties: NAM, an alkaline crystalline and highly water-soluble substance, led to a premature release from conventional shellac microcapsules, whereas RMSD and NA with their medium solubility and neutral to acidic pH properties delayed the shellac dissolution. A precise modulation of the release profile of each substance was possible by the addition of different intermediate subcoatings: an acidic layer with citric acid counteracted the premature release of the alkaline and highly soluble NAM. In contrast, an alkaline sodium bicarbonate intermediate subcoating enhanced shellac swelling and delayed the release of NA and RMSD. In conclusion, the novel triple-layer shellac coating provides a much higher adaptability and reliability for nutritional formulations aiming at a targeted release in the ileocolonic region.


Assuntos
Preparações de Ação Retardada/química , Resinas Vegetais/química , Colo , Liberação Controlada de Fármacos , Mononucleotídeo de Flavina/química , Concentração de Íons de Hidrogênio , Íleo , Niacina/química , Niacinamida/química , Bicarbonato de Sódio/química
8.
Mater Sci Eng C Mater Biol Appl ; 100: 11-22, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948045

RESUMO

Two-dimensional transition metal dichalcogenides (TMDCs), such as MoS2 and WS2, have recently emerged as nanomaterials with potential use in biomedicine. An attractive means to favor their interaction with biological media is the use of proper biomolecules as exfoliating/dispersing agents. Here, MoS2 flakes were stabilized with different small functional biomolecules such as adenosine monophosphate (AMP), guanosine monophosphate (GMP) and flavin mononucleotide (FMN) through the strong nucleotide-MoS2 interaction of Lewis acid-base type, rather than just on the weak dispersive and hydrophobic forces commonly associated with the use of many surfactants. The impact of the nucleotide-stabilized MoS2 flakes on the viability and cell proliferation, on the production of intracellular reactive oxygen species (ROS), and on the preosteoblast differentiation process (early stage) has been also evaluated, as well as the incorporation and intracellular localization of the nanomaterials by MC3T3-E1 and Saos-2 cells. The nucleotide-stabilized MoS2 flakes were found to exhibit excellent biocompatibility. Furthermore, their incorporation did not affect the integrity of the cell plasma membrane, which makes them ideal candidates for delivering drug/gene directly into cells. The in vitro cell response of tumor cells to these nanomaterials differs from that of undifferentiated cells, which provides the basis for their potential use in cancer therapy.


Assuntos
Monofosfato de Adenosina/química , Dissulfetos/química , Mononucleotídeo de Flavina/química , Guanosina Monofosfato/química , Molibdênio/química , Nanoestruturas/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Nanoestruturas/toxicidade , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836629

RESUMO

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.


Assuntos
Apoenzimas/genética , Simulação de Dinâmica Molecular , Fósforo-Oxigênio Liases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Apoenzimas/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Di-Hidroxiacetona/química , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/química , Gliceraldeído/química , Humanos , Fósforo-Oxigênio Liases/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por Substrato
10.
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
11.
ACS Appl Mater Interfaces ; 11(8): 8400-8411, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30724070

RESUMO

The development of novel methods to detect mercury is of paramount importance owing to the impact of this metal on human health and the environment. We observed that flavin mononucleotide (FMN) and its helical assembly with a single-walled carbon nanotube (SWNT) selectively bind Hg2+ arising from HgCl2 and MeHgCl. Absorption spectroscopic studies show that FMN preferentially forms a 2:1 rather than a 1:1 complex with Hg2+ at high FMN concentrations. On the basis of the analogy to the thymine-Hg-thymine complex, it is proposed that the 2:1 complex between FMN and Hg2+ comprises a Hg-bridged pair of FMN groups, regardless of the presence of SWNT. Upon addition of as little as a few hundred nanomoles of Hg2+, both FMN and FMN-SWNT exhibit absorption and photoluminescence (PL) changes. Moreover, FMN-SWNT displays simultaneous multiple sigmoidal changes in PL of SWNT tubes having different chiral vectors. Assessment of binding affinities using the Hill equation suggests that 2:1 and 1:1 complexes form between Hg2+ and FMN groups on the FMN-SWNT. Theoretical calculations indicate that optical changes of the FMN-SWNT originate from Hg-mediated conformational changes occurring on the helical array of FMN on the SWNT. High-resolution transmission electron microscopy revealed that the presence of Hg2+ in complexes with the FMN-SWNT enables visualization of helical periodic undulation of FMN groups along SWNT without the need for staining. Circular dichroism (CD) study revealed that FMN-SWNT whose CD signal mainly originates from FMN decreases dichroic bands upon the addition of Hg2+ owing to the formation of a centrosymmetric FMN-Hg-FMN triad on SWNT. The binding mode specificity and multimodal changes observed in response to Hg2+ ions suggest that systems based on FMN-SWNT can serve as in vivo NIR beacons for the detection of various mercury derivatives.


Assuntos
Mononucleotídeo de Flavina/química , Mercúrio/química , Nanotubos de Carbono/química , Dicroísmo Circular , Teoria da Densidade Funcional , Cloreto de Mercúrio/química , Compostos de Metilmercúrio/química , Microscopia Eletrônica de Transmissão , Espectroscopia de Infravermelho com Transformada de Fourier , Timina/química
12.
Phys Chem Chem Phys ; 21(22): 11589-11598, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30801593

RESUMO

Biodegradation is a cost-effective and environmentally friendly alternative to removing 2,4,6-trinitrotoluene (TNT) pollution. However, mechanisms of TNT biodegradation have been elusive. To enhance the understanding of TNT biotransformation by the Old Yellow Enzyme (OYE) family, we investigated the crucial first-step hydrogen-transfer reaction by molecular dynamics simulations, docking technologies and empirical valence bond calculations. We revealed the significance of the π-π stacking conformation between the substrate TNT and the reduced flavin mononucleotide (FMNH2) cofactor, which is a prerequisite for the aromatic ring reduction of TNT. Under the π-π stacking conformation, the barrier of the hydrogen-transfer reaction in the aromatic ring reduction is about 16 kcal mol-1 lower than that of nitro group reduction. Then, we confirmed the mechanism of controlling the π-π stacking, that is, the π-π interaction competition mechanism. It indicates that the π-π stacking of TNT and FMNH2 occurs only when the π-π interaction between FMNH2 and TNT is stronger than that between TNT and several key residues with aromatic rings. Finally, based on the competition mechanism, the formation of π-π stacking of TNT and FMNH2 can be successfully enabled by removing the aromatic ring of those key residues in enzymes that originally only transform TNT through the nitro group reduction. This testified the validity of the π-π interaction competition mechanism. This work theoretically clarifies the molecular mechanism of the first-step hydrogen-transfer reaction for the biotransformation of TNT by the OYE family. It is helpful to obtain the enzymes that can biodegrade TNT through the aromatic ring reduction.


Assuntos
Flavoproteínas/metabolismo , NADPH Desidrogenase/metabolismo , Trinitrotolueno/metabolismo , Animais , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biotransformação , Domínio Catalítico , Mononucleotídeo de Flavina/química , Flavoproteínas/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Himenópteros/enzimologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NADPH Desidrogenase/química , Oxirredução , Ligação Proteica , Saccharomyces/enzimologia , Eletricidade Estática , Trinitrotolueno/química
13.
Protein Sci ; 28(1): 167-175, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30246917

RESUMO

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD+ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD+ binds in a folded conformation at the interface of the TIM-barrel domain and the extended domain of the enzyme. Comparison of the enzyme-NAD+ structure with that of the ligand-free enzyme revealed a different conformation of a short loop (75-86) that is part of the NAD+ -binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD+ within the binding pocket. An interrupted helix consisting of two α-helices connected by a small three-residue loop binds the pyrophosphate moiety of NAD+ . The adenine moiety of NAD+ appears to π-π stack with Y261. Steric constraints between the adenosine ribose of NAD+ , P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.


Assuntos
Proteínas de Bactérias/química , Mononucleotídeo de Flavina/química , NAD(P)H Desidrogenase (Quinona)/química , NAD/química , Pseudomonas aeruginosa/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Estrutura Secundária de Proteína , Especificidade por Substrato
14.
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
15.
Colloids Surf B Biointerfaces ; 173: 226-232, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30300828

RESUMO

Microemulsions are widely studied as potential ocular drug delivery vehicles. In the present study we show the versatility of possible use microemulsions as ocular delivery vehicle. The ME is loaded with a hydrophilic drug, riboflavin phosphate (RFP) and a lipophilic, docosahexaenoic acid in triglyceride form (TG-DHA), each separately. These drugs treat keratoconus and dry eye syndrome, respectively. The advantage of using ME loaded with RFP is in overcoming eye epithelium debridement during collagen cross-linking therapy for treatment of keratoconus. ME loaded with lipophilic TG-DHA provides convenient dosage in liquid aqueous form of administration of highly lipophilic TG-DHA, which is known as a protective molecule in dry eye syndrome. The capability of RFP-loaded MEs was demonstrated in terms of improvement of biomechanical strength of the rabbit cornea, as a result of successful penetration of RFP through the intact epithelium. TG-DHA-loaded microemulsion applied topically onto an eye with induced dry eye syndrome showed the significant relief of the dry eye condition.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Síndromes do Olho Seco/tratamento farmacológico , Mononucleotídeo de Flavina/farmacologia , Ceratocone/tratamento farmacológico , Triglicerídeos/farmacologia , Animais , Fenômenos Biomecânicos , Colágeno/química , Colágeno/efeitos da radiação , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/química , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/fisiopatologia , Emulsões , Epitélio Anterior/efeitos dos fármacos , Epitélio Anterior/metabolismo , Mononucleotídeo de Flavina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ceratocone/metabolismo , Ceratocone/fisiopatologia , Masculino , Permeabilidade , Coelhos , Triglicerídeos/química , Raios Ultravioleta
16.
Protein Sci ; 28(1): 135-149, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30207005

RESUMO

Lactate monooxygenase (LMO) catalyzes the FMN-dependent "coupled" oxidation of lactate and O2 to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme-bound intermediates. Other α-hydroxy acid oxidase family members follow an "uncoupled pathway," wherein the α-keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of Mycobacterium smegmatis wild-type LMO and a wild-type-like C203A variant at 2.1 Å and 1.7 Å resolution, respectively. The overall LMO fold and active site organization, including a bound sulfate mimicking substrate, resemble those of other α-hydroxy acid oxidases. Based on structural similarity, LMO is similarly distant from lactate oxidase, glycolate oxidase, mandelate dehydrogenase, and flavocytochrome b2 and is the first representative enzyme of its type. Comparisons with other α-hydroxy acid oxidases reveal that LMO has a longer and more compact folded active site loop (Loop 4), which is known in related flavoenzymes to undergo order/disorder transitions to allow substrate/product binding and release. We propose that LMO's Loop 4 has an enhanced stability that is responsible for the slow product release requisite for the coupled pathway. We also note electrostatic features of the LMO active site that promote substrate binding. Whereas the physiological role of LMO remains unknown, we document what can currently be assessed of LMO's distribution in nature, including its unexpected occurrence, presumably through horizontal gene transfer, in halophilic archaea and in a limited group of fungi of the genus Beauveria. BROAD STATEMENT OF IMPACT: This first crystal structure of the FMN-dependent α-hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its "coupled" oxidation of lactate and O2 to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.


Assuntos
Proteínas de Bactérias/química , Mononucleotídeo de Flavina/química , Oxigenases de Função Mista/química , Mycobacterium smegmatis/enzimologia , Domínio Catalítico , Cristalografia por Raios X
17.
Protein Sci ; 28(1): 123-134, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30171650

RESUMO

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.


Assuntos
Proteínas de Bactérias/química , FMN Redutase/química , Mononucleotídeo de Flavina/química , Mutação de Sentido Incorreto , NADP/química , Multimerização Proteica , Pseudomonas aeruginosa/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , FMN Redutase/genética , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética
18.
Chemphyschem ; 20(3): 405-409, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30417568

RESUMO

Bacterial bioluminescence is initiated by the oxygenation reaction of reduced flavin mononucleotide in luciferase. This enzymatic oxygenation occurs in a wide range of biological processes including cellular redox metabolism, biocatalysis, biosynthesis and homeostasis. However, little is known about the mechanism of the enzymatic reaction between singlet reduced flavin and triplet oxygen. To explore the enigmatic oxygenation, for the first time, the reaction of reduced flavin anion with oxygen was studied in bacterial luciferase by a combined quantum mechanics and molecular mechanics method as well as molecular dynamics simulation. The calculated results demonstrate that the reaction proceeds via a proton-coupled electron transfer (PCET) pathway, and the essential αHis44 acts as a catalytic acid to provide the proton. The currently proposed PCET mechanism clearly describes the initial steps of bacterial bioluminescence, and could be suitable for the other flavin oxygenation reactions in enzymes.


Assuntos
Mononucleotídeo de Flavina/química , Luciferases/química , Vibrio/enzimologia , Transporte de Elétrons , Luminescência , Medições Luminescentes , Simulação de Dinâmica Molecular , Oxirredução , Oxigênio/química , Prótons , Vibrio/química
19.
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
20.
J Am Chem Soc ; 140(48): 16521-16527, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30412389

RESUMO

Until now, FMN/FAD radicals could not be stabilized in aqueous solution or other protic solvents because of rapid and efficient dismutation reactions. In this contribution, a novel system for stabilizing flavin radicals in aqueous solution is reported. Subsequent to trapping FMN in an agarose matrix, light-generated FMN radicals could be produced that were stable for days even under aerobic conditions, and their concentrations were high enough for extensive EPR characterization. All large hyperfine couplings could be extracted by using a combination of continuous-wave EPR and low-temperature ENDOR spectroscopy. To map differences in the electronic structure of flavin radicals, two exemplary proton hyperfine couplings were compared with published values from various neutral and anionic flavoprotein radicals: C(6)H and C(8α)H 3. It turned out that FMN•- in an aqueous environment shows the largest hyperfine couplings, whereas for FMNH• under similar conditions, hyperfine couplings are at the lower end and the values of both vary by up to 30%. This finding demonstrates that protein-cofactor interactions in neutral and anionic flavoprotein radicals can alter their electron spin density in different directions. With this aqueous system that allows the characterization of flavin radicals without protein interactions and that can be extended by using selective isotope labeling, a powerful tool is now at hand to quantify interactions in flavin radicals that modulate the reactivity in different flavoproteins.


Assuntos
Mononucleotídeo de Flavina/química , Radicais Livres/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Flavoproteínas/química , Géis/química , Sefarose/química , Água/química
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