Armand W. J. W. Tepper

Shape and release control of a peptide decorated vesicle through pH sensitive orthogonal supramolecular interactions.

A pH sensitive carrier is obtained by coating a cyclodextrin vesicle with an adamantane-terminated octapeptide through the formation of an inclusion complex. Upon lowering the pH from 7.4 to 5.0, the formation of peptide beta-sheets on the vesicle surface induces a transition of the bilayer from a sphere to a fiber. This transition is fully reversible and repeatable. The vesicles release their cargo upon fiber formation.

1H NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase

The 600 MHz 1H NMR spectrum of tyrosinase (31 kDa) of Streptomyces antibioticus in the oxidized, chloride‐bound form is reported. The downfield part of the spectrum (15–55 ppm) exhibits a large number of paramagnetically shifted signals. The paramagnetism is ascribed to a thermally populated triplet state. The signals derive from six histidines binding to the metals through their Nϵ atoms. There is no evidence for endogenous bridges. The exchange coupling, −2J, amounts to 298 cm−1. In the absence of chloride the peaks broaden. This is ascribed to a slowing down of the electronic relaxation. The exchange coupling decreases to −2J=103 cm−1.

Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase

Abstract A scaled-up isolation and purification procedure is described for tyrosinase from Streptomyces antibioticus . Kinetic studies of the enzyme catalysed conversion of l -3,4-dihydroxyphenylalanine ( l -DOPA) into DOPAchrome show that kojic acid, l -mimosine, p -toluic acid and benzoic acid exhibit competitive inhibition with inhibition constants of 3.4, 30, 1.9×10 2 and 8.0×10 2 μM, respectively. Paramagnetic NMR techniques appear well suited to study the binding of inhibitors to the active site. From the variation of the NMR shifts with temperature a value of −2 J =156±6 cm −1 is derived for the exchange coupling between the unpaired spins on the two Cu(II) ions in the active site of the met-tyrosinase/kojic acid complex.

Involvement of Tyr108 in the enzyme mechanism of the small laccase from Streptomyces coelicolor.

The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cus in the trinuclear center, as noted before, but also to the enzyme mechanism.

Site−Site Interactions Enhances Intramolecular Electron Transfer in Streptomyces coelicolor laccase

Control of electron transfer rates, caused by intrinsic protein structural properties, is an intriguing feature of internal biological electron transfer (ET) reactions. The small laccase (SLAC) isolated from Streptomyces coelicolor has recently been shown to have structural and reactivity features distinct from those of other laccases. While other copper oxidases contain three cupredoxin domains, the SLAC 3D structure has recently been determined and shown to consist of only two, and a different reaction intermediate has been reported for it. It was therefore of particular interest to investigate the intramolecular ET between the type 1 and the trinuclear copper center in SLAC which is a crucial step in the catalytic cycle of the multicopper oxidases, leading to dioxygen reduction to water. This ET step was found to markedly depend on the reduction state of the enzyme, possibly reflecting site-site interactions so far not observed in other multicopper oxidases.

Identification of a Radical Intermediate in the Enzymatic Reduction of Oxygen by a Small Laccase

The enzyme mechanism of the Cu-containing small laccase (SLAC) from Streptomyces coelicolor has been investigated by optical and electron paramagnetic resonance spectroscopy. A new intermediate was identified after the reaction of molecular oxygen with the reduced trinuclear site of the type-1-depleted (T1D) form of the enzyme. It has the fingerprint of a biradical with a triplet ground state. One of the spins resides on a Cu in the trinuclear site, tentatively identified as the type-2 site, while the other spin derives from a protein-based radical. The latter is tentatively identified as a tyrosyl radical on the basis of the similarity of the optical characteristics with those observed for a Cu tyrosyl radical pair. The spin-spin distance was found to be 5.0 +/- 0.2 A.

Electrical Contacting of an Assembly of Pseudoazurin and Nitrite Reductase Using DNA-Directed Immobilization

A method for the electrical contacting of redox enzymes that obtain oxidizing or reducing equivalents from small electron-transfer proteins is demonstrated. The electrochemical contacting of redox enzymes through their immobilization onto electrode supports offers great potential for technological applications and for fundamental studies, but finding appropriate methods to immobilize the enzymes in an orientation allowing rapid electron transfer with the electrode has proven difficult. The copper enzyme nitrite reductase (NiR) and its natural electron-exchange partner pseudoazurin (Paz) are conjugated to a specific DNA tag and immobilized to a gold electrode into a stoichiometrically defined assembly. The DNA tethered to the electrode surface acts as flexible place-holder for the protein components, allowing both proteins to move within the construct. It is shown that Paz efficiently shuttles electrons between the electrode and the NiR enzyme, allowing the electrochemically driven NiR catalysis to be monitored. The activity of the NiR enzyme remains unperturbed by the immobilization. The rate-limiting step of the system is tentatively ascribed to the dissociation of the Paz/NiR complex. The electrochemical response of the system reports not only on the NiR catalysis and on interfacial electron transfer but also on the interaction between NiR and Paz.

A protein-based oxygen biosensor for high-throughput monitoring of cell growth and cell viability

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.

Sensitive detection of histamine using fluorescently labeled oxido-reductases

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.

Channeling of electrons within SLAC, the small laccase from Streptomyces coelicolor

The reduction kinetics of the fluorescently labeled small laccase (SLAC) from Streptomyces coelicolor was 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 k(red) = 0.28 +/- 0.02 microM(-1/2) s(-1).