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1.
Photochem Photobiol Sci ; 19(2): 159-170, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31922165

RESUMO

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an α-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of ≈10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) × 105 M-1 s-1, and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) × 10-6 M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Rhodobacter sphaeroides/química , Corantes/química , Dimerização , Flavinas/química , Cinética , Luz , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Rhodobacter sphaeroides/metabolismo
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117346, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31344577

RESUMO

The photophysics and structural transition dynamics of a bio-active flavin lumichrome (LM) entrapped in two sugars based neutral surfactants were reported. Sugar-based surfactants, which were used for research purpose are potential environmentally friendly, green alternative amphiphilic surface active substance compared to other kinds of common surfactants. Here, two alkyl glucoside surfactants n-octyl-ß-D-glucopyranoside (OBG) and n-octyl-ß-D-thioglucopyranoside (OBTG) were used. This work is carried out by using steady-state absorption and fluorescence emission spectroscopy along with time-resolved fluorescence emission techniques. Photophysics of LM was modulated several folds in these two sugar-based neutral micelles. LM exhibits excitation and emission wavelength dependent fluorescence properties in these two sugars based neutral micelles. LM confined in the micellar environments exhibited structural transition dynamism, i.e. different kinds of conformers are equilibrated. Herein, different conformers of LM are identified and explained with the help of spectroscopic methods. From the fluorescence anisotropy measurement, it was found that the rotational relaxation time of LM in OBG micelle was more compared to that in OBTG micelle, which indicates that the LM molecule faced much more constrained environment in OBG micellar media.


Assuntos
Flavinas/química , Glucosídeos/química , Tensoativos/química , Tioglucosídeos/química , Flavinas/análise , Micelas , Modelos Moleculares , Espectrometria de Fluorescência
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117399, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31344575

RESUMO

In this study, we analyzed some monofunctional Ru (II) complexes containing chlorine, bromine and fluorine atoms around the central atom. The best calculation level among HF, B3LYP and M062X methods for [Ru (Cl-Ph-tpy)(NN)X]+ (X = F, Cl, Br) was determined in the light of Benchmark analysis and according to this analysis results, the best level is shown as B3LYP-LANL2DZ/6-31G(d). In addition to this, the spectroscopic data (IR, NMR and UV-Vis) were also obtained in agreement with experimental results. The tendency of anticancer activity and structural activity relationship (SAR) parameters are predicted with some quantum chemical methods. Surface and contour diagrams, as well as electron densities on mentioned complexes were interpreted through theoretically obtained results. Finally, the anticancer activity tendency of the relevant complexes on the human cervical carcinoma cell line (ID: 1 M17) is supported by molecular docking calculations.


Assuntos
Flavinas/química , Glucosídeos/química , Rutênio/química , Tensoativos/química , Tioglucosídeos/química , Micelas , Simulação de Acoplamento Molecular , Espectrometria de Fluorescência
4.
Biochemistry (Mosc) ; 84(11): 1411-1423, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760927

RESUMO

Ischemic stroke and neonatal hypoxic-ischemic encephalopathy are two of the leading causes of disability in adults and infants. The energy demands of the brain are provided by mitochondrial oxidative phosphorylation. Ischemia/reperfusion (I/R) affects the production of ATP in brain mitochondria, leading to energy failure and death of the affected tissue. Among the enzymes of the mitochondrial respiratory chain, mitochondrial complex I is the most sensitive to I/R; however, the mechanisms of its inhibition are poorly understood. This article reviews some of the existing data on the mitochondria impairment during I/R and proposes two distinct mechanisms of complex I damage emerging from recent studies. One mechanism is a reversible dissociation of natural flavin mononucleotide cofactor from the enzyme I after ischemia. Another mechanism is a modification of critical cysteine residue of complex I involved into the active/deactive conformational transition of the enzyme. I describe potential effects of these two processes in the development of mitochondrial I/R injury and briefly discuss possible neuroprotective strategies to ameliorate I/R brain injury.


Assuntos
Encéfalo/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Flavinas/química , Flavinas/metabolismo , Humanos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Compostos de Sulfidrila/química
5.
Microbiol Res ; 229: 126324, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31491671

RESUMO

Through extracellular electron transfer (EET), bacteria are capable of transforming different insoluble materials of geochemical interest into energy-rich molecules for their growth. For this process, bacteria have been depending directly or indirectly on molecules synthesized within the cells or by various synthetics as mediators. Herein, we studied the in-situ change in electrochemistry and supporting components for EET in the extracellular polysaccharide (EPS) producing biofilm of thermophilic Geobacillus sp. The CV and DPV resultsrevealed that the intact biofilm of bacteria was not able to generate any potential at 25 °C /- ≤50 °C. However, at 55 °C (optimal condition), the potential occurred drastically after the EPS production by bacteria. HPLC and MALDI-TOF results revealed that the presence of Flavins, which can able adsorbed to the electrodes from the cell surface. Moreover, the temperature-dependent EPS production and originally conceived ability of flavins to act as electron shuttles suggest that not much complexity in bacteria with minerals. Additionally, the electrochemical potential was severely affected upon removal of EPS/flavin moiety from the intact biofilm, revealed the necessity of EPS bound flavins in transferring the electrons across its thick cell walls. This paradigm shift to electrogenic nature of Geobacillus sp. biofilm will become evident in the adaptation of other microbes during mineral respiration in extreme environments.


Assuntos
Flavinas/metabolismo , Geobacillus/metabolismo , Polissacarídeos Bacterianos/metabolismo , Biofilmes , Transporte de Elétrons , Elétrons , Flavinas/química , Geobacillus/química , Geobacillus/genética , Polissacarídeos Bacterianos/química
6.
J Microbiol Biotechnol ; 29(10): 1603-1606, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474099

RESUMO

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Staphylococcus aureus/patogenicidade , Streptomyces/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Flavinas/química , Flavinas/isolamento & purificação , Flavinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Mutação , Staphylococcus aureus/enzimologia , Streptomyces/metabolismo , Virulência/efeitos dos fármacos
7.
J Photochem Photobiol B ; 198: 111546, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31351309

RESUMO

Fluorescence dynamics of human d-amino acid oxidase (hDAAO) and its five inhibitors have been studied in the picoseconds time domain, and compared with one in d-amino acid oxidase from porcine kidney (pkDAAO) reported. The fluorescence lifetimes were identified as 47 ps in the dimer, 235 ps in the monomer, which are compared with those of pkDAAO (45 ps-185 ps). The fluorescence lifetimes of the hDAAO did not change upon the inhibitor bindings despite of modifications in the absorption spectra. This indicates that the lifetimes of the complexes are too short to detect with the picosecond lifetime instrument. Numbers of the aromatic amino acids are similar between the both DAAOs. The fluorescence lifetimes of hDAAO were analysed with an ET theory using the crystal structure. The difference in the lifetimes of the dimer and monomer was well described in terms of difference in the electron affinity of the excited isoalloxazine (Iso*) between the two forms of the protein, though it is not known whether the structure of the monomer is different from the dimer. Three fastest ET donors were Tyr314, Trp52 and Tyr224 in the dimer, while Tyr314, Tyr224 and Tyr55 in the monomer, which are compared to those in pkDAAO, Tyr314, Tyr224 and Tyr228 in the dimer, and Tyr224, Tyr314 and Tyr228 in the monomer. The ET rate from Trp55 in hDAAO dimer was much faster compared to the rate in pkDAAO dimer. A rise component with negative pre-exponential factor was not observed in hDAAO, which are found in pkDAAO.


Assuntos
Aminoácidos Aromáticos/química , D-Aminoácido Oxidase/química , Flavinas/química , Rim/enzimologia , Animais , D-Aminoácido Oxidase/metabolismo , Dimerização , Transporte de Elétrons , Humanos , Ligação de Hidrogênio , Espectrometria de Fluorescência , Eletricidade Estática , Suínos
8.
Phys Chem Chem Phys ; 21(25): 13453-13461, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31187821

RESUMO

Radical pair formation and decay are implicated in a wide range of biological processes including avian magnetoreception. However, studying such biological radical pairs is complicated by both the complexity and relative fragility of natural systems. To resolve open questions about how natural flavin-amino acid radical pair systems are engineered, and to create new systems with novel properties, we developed a stable and highly adaptable de novo artificial protein system. These protein maquettes are designed with intentional simplicity and transparency to tolerate aggressive manipulations that are impractical or impossible in natural proteins. Here we characterize the ultrafast dynamics of a series of maquettes with differing electron-transfer distance between a covalently ligated flavin and a tryptophan in an environment free of other potential radical centers. We resolve the spectral signatures of the cysteine-ligated flavin singlet and triplet states and reveal the picosecond formation and recombination of singlet-born radical pairs. Magnetic field-sensitive triplet-born radical pair formation and recombination occurs at longer timescales. These results suggest that both triplet- and singlet-born radical pairs could be exploited as biological magnetic sensors.


Assuntos
Flavinas/química , Proteínas/química , Triptofano/química , Cisteína/química , Transporte de Elétrons , Radicais Livres/química , Cinética , Campos Magnéticos , Modelos Moleculares , Oxirredução
9.
Mar Drugs ; 17(6)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242631

RESUMO

Previously unreported meroterpene, acremine S (1), and benzopyran derivative, acremine T (2), were isolated, together with lumichrome (3), ergosterol (4) and ergosterol 5,8-endoperoxide, from cultures of the marine sponge-associated fungus Acremonium persicinum KUF1007. The structure of the previously unreported compounds was established based on an extensive analysis of 1D and 2D NMR spectra as well as HRMS data. The absolute configurations of the stereogenic centers of 1 were established, unambiguously, based on NOESY correlations and comparison of calculated and experimental electronic circular dichroism (ECD) spectra. Compounds 1-3 were tested for their in vitro acetylcholinesterase and butyrylcholinesterase inhibitory activities.


Assuntos
Acremonium/química , Benzofuranos/química , Inibidores da Colinesterase/química , Poríferos/microbiologia , Terpenos/química , Animais , Dicroísmo Circular/métodos , Ergosterol/química , Flavinas/química , Espectroscopia de Ressonância Magnética/métodos , Testes de Sensibilidade Microbiana/métodos
10.
ACS Chem Biol ; 14(6): 1110-1114, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31091068

RESUMO

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 "poisons". Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.


Assuntos
Compostos Aza/química , Proteínas de Ligação a DNA/antagonistas & inibidores , Etoposídeo/farmacocinética , Flavinas/farmacologia , Inibidores da Topoisomerase II/farmacocinética , Animais , Transporte Biológico , Linhagem Celular , Galinhas , Flavinas/química , Flavinas/farmacocinética , Humanos , Diester Fosfórico Hidrolases , Bibliotecas de Moléculas Pequenas/farmacologia
11.
Methods Enzymol ; 620: 1-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072483

RESUMO

A key factor for flavoenzyme activity is the reduction potential of the bound flavin. The reduction potentials of protein-bound flavins span approximately a 500-mV range consistent with flavoenzymes having critical roles in metabolism and a variety of biological processes. Redox potentials of flavoenzymes have traditionally been determined using an electrode-based system with either direct or indirect electrochemical coupling between the protein and the working electrode. An electrode independent method, however, is also now commonly used and involves calculating the unknown flavin reduction potential of the protein from the known reduction potential of a reference or indicator dye. Here, the "classic" potentiometric method and the xanthine/xanthine oxidase methods are described. Both methods rely on equilibrium between protein-bound flavin and redox dyes. The potentiometric method measures the equilibrated redox potential of the protein-dye mixture whereas the xanthine/xanthine oxidase technique relies on slow continuous enzymatic reduction to maintain a constant equilibrium between the protein and the dyes. Because electrochemical equipment is not required, the xanthine/xanthine oxidase method is more accessible and convenient for researchers seeking to determine reduction potentials of flavoproteins or other biological redox centers such as hemes. The xanthine/xanthine oxidase method has been used to determine flavin reduction potentials from +132 to -417mV, demonstrating it is suitable for characterizing the redox properties of most flavoproteins.


Assuntos
Ensaios Enzimáticos/métodos , Benzil Viologênio/química , Flavinas/química , Flavoproteínas/química , Indicadores e Reagentes/química , Oxirredução , Paraquat/química , Potenciometria/métodos , Xantina/química , Xantina Oxidase/química
12.
Methods Enzymol ; 620: 115-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072484

RESUMO

This account describes the application of kinetic isotope effects (KIEs) to investigate the mechanistic properties of flavin dependent enzymes. Assays can be conducted during steady-state catalytic turnover of the flavoenzyme with its substrate or by using rapid-kinetic techniques to measure either the reductive or oxidative half-reactions of the enzyme. Great care should be taken to ensure that the observed effects are due to isotopic substitution and not other factors such as pH effects or changes in the solvent viscosity of the reaction mixture. Different types of KIEs are described along with a physical description of their origins and the unique information each can provide about the mechanism of an enzyme. Detailed experimental techniques are outlined with special emphasis on the proper controls and data analysis that must be carried out to avoid erroneous conclusions. Examples are provided for each type of KIE measurement from references in the literature. It is our hope that this article will clarify any confusion concerning the utility of KIEs in the study of flavoprotein mechanism and encourage their use by the community.


Assuntos
Ensaios Enzimáticos/métodos , Enzimas/química , Flavoproteínas/química , Biocatálise , Flavinas/química , Concentração de Íons de Hidrogênio , Isótopos/química , Cinética , Oxirredução
13.
Methods Enzymol ; 620: 167-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072486

RESUMO

Flavoenzymes mediate a large number of different chemical transformations that employ the flavin coenzyme for many different purposes. Flavins are commonly utilized in a variety of both 1- and 2-electron transfers and reactions involving oxygen activation. In addition, flavins have also been shown to function as acid/base catalysts or nucleophilic catalysts, to electrostatically stabilize transition states, and to serve simply as structural components in some enzymes. In all of these functions, the electronic properties of the flavin play a critical role. Studies carried out over a number of years have shown that these electronic properties (and subsequently, the catalytic properties of the flavoenzyme) can be manipulated by altering the substituents on the isoalloxazine ring system of the flavin. Here, we detail methods for the chemoenzymatic preparation and purification of flavin analogues, the reconstitution of apo-flavoenzymes with these analogues, and the use of linear free energy relationships (LFERs) to correlate the perturbations induced by the altered substituents. Using examples from the literature, we highlight how the use of flavin analogues and LFERs can provide mechanistic insight into the transition state structures that define flavoenyzme chemical mechanisms.


Assuntos
Ensaios Enzimáticos/métodos , Enzimas/química , Flavoproteínas/química , Flavinas/química , Cinética , Oxirredução , Termodinâmica
14.
Methods Enzymol ; 620: 189-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072487

RESUMO

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.


Assuntos
Enzimas/química , Flavoproteínas/química , Análise Espectral Raman/métodos , Flavinas/química , Vibração
15.
Methods Enzymol ; 620: 215-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072488

RESUMO

The optical spectrum of a flavoprotein is one of its signature properties. No two flavoprotein absorption spectra are exactly alike as each encodes the details of the interaction of the flavin cofactor electronic structure with the specific protein binding pocket. Electronic Stark spectroscopy has the potential to elucidate these interactions with high sensitivity, at low cost, and requiring minimal technical sophistication. In this chapter we will outline the theoretical basis for Stark spectroscopy and describe the construction of the Stark spectrometer. Step-by-step instructions are given for acquiring and interpreting Stark spectra to retrieve difference moments of the flavin ground versus excited state charge distributions.


Assuntos
Enzimas/química , Flavoproteínas/química , Análise Espectral/métodos , Absorção Fisico-Química , Elétrons , Flavinas/química , Oxirredução
16.
Methods Enzymol ; 620: 277-314, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072491

RESUMO

The general field of molecular simulation provides a wide spectrum of methods for studying the structure and function of biomolecules. Depending on the scale and question of interest, appropriate approaches may range from ab initio quantum mechanical calculations (when detailed aspects of and changes in electronic structure must be considered) to Brownian dynamics and coarse-grained molecular dynamics (to track large scale conformational motions, diffusion, and inter-molecular interactions). The entire range of molecular simulation methods has been fruitfully applied to a range of flavoenzymes, allowing researchers to address everything from the specific intermediates involved in the photoreactions of flavin chromophore-containing light sensors, to the very long timescale motions induced by covalent modifications to bound flavin. The unique challenge posed by flavoproteins to all types of molecular simulation arises from the chemistry of the flavin isoalloxazine moiety, which presents an unusually large delocalized electron system which must be carefully treated in order to represent its contributions to the overall behavior of the system. Here we outline the particular considerations required for appropriate treatment of flavoproteins in simulations ranging from electronic structure calculations to long-timescale modeling of flavoprotein conformational transitions.


Assuntos
Flavoproteínas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Flavinas/química , Conformação Molecular
17.
Methods Enzymol ; 620: 349-363, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072493

RESUMO

As a rare feature among organic cofactors, reduced flavins (Flred) can efficiently react with dioxygen (O2). As a consequence, many flavin-dependent enzymes may serve as either oxidases that use O2 as an electron acceptor or as monooxygenases that transfer one oxygen atom derived from O2 to an organic substrate. For the latter functionality, covalent flavin: oxygen adducts are formed that function as oxygenating species. Remarkably, despite intensive research, many open questions remain how flavoenzymes control the reaction with O2. Here, we describe O2-pressurized protein crystallography in detail as a structural approach to gain insight into the interactions between the protein scaffold, the flavin cofactor and O2. This may allow to further our understanding of how flavoenzymes can steer the formation of different oxygenating species and thus provide missing puzzle pieces for rational flavoenzyme design.


Assuntos
Cristalografia por Raios X/métodos , Ensaios Enzimáticos/métodos , Flavinas/química , Flavoproteínas/química , Oxirredução , Oxirredutases/química , Oxigênio/química
18.
Methods Enzymol ; 620: 365-398, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072494

RESUMO

Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called "flavin-based electron bifurcation." The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins.


Assuntos
Flavoproteínas Transferidoras de Elétrons/química , Ensaios Enzimáticos/métodos , Transporte de Elétrons , Ensaios Enzimáticos/instrumentação , Flavinas/química , Modelos Moleculares , NADP/química , Oxirredução
19.
Methods Enzymol ; 620: 399-422, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072495

RESUMO

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.


Assuntos
Ensaios Enzimáticos/métodos , FMN Redutase/química , Oxigenases de Função Mista/química , Flavinas/química , Cinética , Oxirredução , Ligação Proteica , Especificidade por Substrato
20.
Methods Enzymol ; 620: 455-468, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072497

RESUMO

Flavin-N5-oxide is a new intermediate in flavoenzymology. Here we describe the identification of DszA (dibenzothiophene catabolism), RutA (uracil catabolism) and HcbA1 (hexachlorobenzene catabolism) as flavin-N5-oxide-utilizing enzymes. Mechanistic analysis of these reactions suggests a model for the identification of other examples of this catalytic motif.


Assuntos
Proteínas de Bactérias/química , Ensaios Enzimáticos/métodos , Flavinas/química , Oxigenases/química , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Escherichia coli , Hexaclorobenzeno/química , Oxigenases/isolamento & purificação , Rhodococcus , Tiofenos/química , Uracila/química
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