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The interaction of the monooxygenating type -3 copper enzyme Tyrosinase (Ty) from Streptomyces antibioticus with its inhibitor p-nitrophenol (pnp) was studied by paramagnetic NMR methods. The relevance of the experimental findings toward the Ty catalytic mechanism is discussed.

While it is generally accepted that α‐synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA‐stabilized human αSOs as a bait protein. The protocol involved co‐immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo‐LC‐SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs‐binding partners. We identify in total 29 proteins associated with DHA‐αSO of which eleven are membrane proteins, including synaptobrevin‐2B (VAMP‐2B), the sodium–potassium pump (Na+/K+ ATPase), the V‐type ATPase, the voltage‐dependent anion channel and calcium‐/calmodulin‐dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab‐3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot‐blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease‐modifying treatments targeting toxic αSOs. [ABSTRACT FROM AUTHOR]

A critical step in Parkinson's disease (PD) is the formation of toxic α-synuclein oligomers (αSOs). In vitro αSOs are formed by self-assembly of α-synuclein at high concentrations, or by the addition of, for example, dopamine, lipids, ethanol, or metal ions. These αSOs are structurally distinct from the unfolded monomer and aggregated β-sheet fibrils. Nevertheless, the literature reports a wide variety of αSO shapes, sizes, and proposed toxic mechanisms. This heterogeneous character makes it difficult to form a unifying picture. Here, we present an overview of the different αSO species made in vitro, providing a tool for better comparison of different protocols and the ensuing αSOs, and emphasizing the striking versatility in the appearance and properties of these critical species. We also summarize what is known of the biological activities of different αSOs. Despite a large and increasing level of insight into αSO effects in vitro, we still lack strong insight into the structures and sizes of αSO species formed in vivo. Once this is established, it may be possible to generate more uniform protocols that could stimulate further efforts to develop viable PD biomarker assays and therapies. [ABSTRACT FROM AUTHOR]

The reduction kinetics of the fluorescently labeled small laccase (SLAC) from Streptomyces coelicolorwas studied by stopped flow kinetic measurements. The tryptophan fluorescence and the emission from a covalently attached label were used to selectively follow the progress of the reduction of the trinuclear copper center (TNC) and the type-1 (T1) Cu site in the enzyme as a function of time. A numerical analysis of the kinetic traces provided new insight into the midpoint potential difference between the T1 and the TNC site as the TNC becomes stepwise charged with electrons. The change in fluorescence of the TNC as the reduction of the TNC proceeds provided evidence that the type-3 dinuclear part of the TNC becomes charged prior to the reduction of the type-2 (T2) center of the TNC. The rate of reduction of the enzyme by dithionite (DT) appeared proportional to the square root of the DT concentration with a rate constant of kred= 0.28 ± 0.02 μM−1/2s−1. [ABSTRACT FROM AUTHOR]

The article presents a research on using a small laccase (SLAC) for enzymatic oxygen reduction. It notes that a transient biradical species of multicopper oxidases which has been distinguished during SLAC turnover is being used in the study. A two-electron oxidation is used in the first step of the oxygen reduction process.

The article presents a study which examines the effect of site-site interactions on the intramolecular electron transfer (ET) rates of Streptomyces coelicolor (SLAC). It states that ET rates depend on the electron enzyme reductions that are observed in multicopper oxidases (MCOs). It adds that the activation parameters of ET rates in SLAC reduction are affected by temperature. It concludes that the internal ET rates control of site-site interactions can be beneficial in reducing substrates.

The article presents a study which demonstrates the release of the encapsulated contents of a decorated cyclodextrin vesicles through the pH sensitive orthogonal supramolecular interactions. In this study, the surface of the cyclodextrin vehicles was decorated with β-sheet domains by synthesizing adamantane modified octapeptide. It was observed that the employment of the secondary structure of the octapeptide results to a reversible change of the supramolecular complex from the vesicles.

While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium-potassium pump (Na + /K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs., (© 2019 Federation of European Biochemical Societies.)

In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluorescent tag, in combination with soluble quinoprotein (PQQ-containing) glucose dehydrogenase (s-GDH) to measure trace amounts of phenols is explored. Proof of concept is provided by a series of experiments, which show a clear quantitative dependence of the response on the phenol concentration. One of the advantages of the detection system is that apart from a standard fluorimeter no further instrumentation is required., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM-225 μM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 μM with a coefficient of variation of 11% for 10 measurements of 100nM histamine in a 100 μL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fluorescently labeled hemocyanin has been previously proposed as an oxygen sensor. In this study, we explored the efficacy of this biosensor for monitoring the biological oxygen consumption of bacteria and its use in testing bacterial cell growth and viability of Escherichia coli, Pseudomonas aeruginosa, Paracoccus denitrificans, and Staphylococcus simulans. Using a microwell plate, the time courses for the complete deoxygenation of samples with different initial concentrations of cells were obtained and the doubling times were extracted. The applicability of our fluorescence-based cell growth assay as an antibacterial drug screening method was also explored. The results provide a proof-of-principle for a simple, quantitative, and sensitive method for high-throughput monitoring of prokaryotic cell growth and antibiotic susceptibility screening.

A fluorescence-based system to sense oxygen in solution is described. The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. The main protein in this study, a recombinant tyrosinase from the soil bacterium Streptomyces antibioticus, has been covalently labeled with a variety of fluorescent dye molecules with emission maxima spanning the whole visible wavelength range. In all cases, the emission of the label varied considerably between O2-bound and O2-free protein with a contrast exceeding that of the Trp emission for some labels. It is shown that different constructs may be simultaneously observed using a single excitation wavelength. Next to the described application in oxygen sensing, the method may be applicable to any protein showing variations in tryptophan fluorescence, for example as a function of ligand binding or catalysis.

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms.

Tyrosinase (Ty) is a type 3 copper protein involved in the rate-limiting step of melanin synthesis. It is shown that the endogenous Trp fluorescence of tyrosinase from Streptomyces antibioticus is remarkably sensitive to the redox state. The fluorescence emission intensity of the [(Cu(I) Cu(I)] reduced species is more than twice that of the oxygen-bound [Cu(II)-O(2)(2-)-Cu(II)] form. The emission intensity of the oxidized [Cu(II)-OH(-)-Cu(II)] protein (Ty(met)) appears to be dependent on an acid-base equilibrium with a pK(a) value of 4.5 +/- 0.1. The binding of fluoride was studied under pseudo first-order conditions using stopped-flow fluorescence spectroscopy. The kinetic parameters k(on), K(d), and the fraction of fluorescence emission quenched upon fluoride binding show a similar pH dependence as above with an average pK(a) value of 4.62 +/- 0.05. Both observations are related to the dissociation of Cu(2)-bridging hydroxide at low pH. It is further shown that Ty is rapidly inactivated at low pH and that halide protects the enzyme from this inactivation. All results support the hypothesis that halide displaces hydroxide as the Cu(2)-bridging ligand in Ty(met). The relevance of the experimental findings for the catalytic cycle is discussed. The data are consistent with the data obtained from other techniques, validating the use of fluorescence quenching as a sensitive and effective tool in studying ligand binding and substrate conversion.

The structural basis that define the physiological functions of binuclear copper enzymes is discussed in the frame of the data generated by a broad spectroscopic approach, spanning from paramagnetic NMR and pulsed EPR to x-ray absorption spectroscopies. The structural features discussed for the different oxidation and ligation states accessible to a binuclear copper sites are the coordination geometry for the first and second shell, the metal-metal distance and the role of the bridging exogenous ligand(s). A structural model will be presented to rationalize both the differentiation in function within the protein families and the reaction mechanism of those proteins that are enzymatically active.