Structural and Functional Consequences Induced by Post-Translational Modifications in α-Defensins

HNP-1 is an antimicrobial peptide that undergoes proteolytic cleavage to become a mature peptide. This process represents the mechanism commonly used by the cells to obtain a fully active antimicrobial peptide. In addition, it has been recently described that HNP-1 is recognized as substrate by the arginine-specific ADP-ribosyltransferase-1. Arginine-specific mono-ADP-ribosylation is an enzyme-catalyzed post-translational modification in which NAD+ serves as donor of the ADP-ribose moiety, which is transferred to the guanidino group of arginines in target proteins. While the arginine carries one positive charge, the ADP-ribose is negatively charged at the phosphate moieties at physiological pH. Therefore, the attachment of one or more ADP-ribose units results in a marked change of cationicity. ADP-ribosylation of HNP-1 drastically reduces its cytotoxic and antibacterial activities. While the chemotactic activity of HNP-1 remains unaltered, its ability to induce interleukin-8 production is enhanced. The arginine 14 of HNP-1 modified by the ADP-ribose is in some cases processed into ornithine, perhaps representing a different modality in the regulation of HNP-1 activities

Streptomyces Secondary Metabolites

Actinobacteria are found spread widely in nature and particular attention is given to their role in the production of various bioactive secondary metabolites. Tests on soil samples show that there can be a diversity of actinomycetes depending on the climate, the area it is growing in, how dry the soil is, and the quality of the soil. However, it was agreed after tests in Yunnan, China, that the genus Streptomyces sp. is most important in ecological function, representing up to 90% of all soil actinomycetes, and therefore helping to show the important characteristics needed of the soil actinomycete population. Streptomycete compounds are used for other biological activities, not just for antibiotics. It has been found that metabolites can be broadly divided into four classes: (1) regulatory activities in compounds, these include consideration of growth factors, morphogenic agents and siderophores, and plants promoting rhizobia; (2) antagonistic agents, these include antiprotozoans, antibacterials, antifungals, as well as antivirals; (3) agrobiologicals, these include insecticides, pesticides, and herbicides; and (4) pharmacological agents, these include neurological agents, immunomodulators, antitumorals, and enzyme inhibitors. It is found that Streptomyces hygroscopicus is one of the very best examples because it secretes in excess of 180 secondary metabolites to locate simultaneous bioactivities for a given compound. Increasingly, both its agricultural and pharmacological screenings are being used in conjunction with antimicrobial tests and have revealed several unusual aerobiological and therapeutic agents, which were hitherto unknown for biological use as antibiotics. Since streptomycetes are now being used increasingly to screen for antimicrobial activity, reports show the existence of secondary metabolites with other activities that may have been missed. Currently, nearly 17% of biologically active secondary metabolites (nearly 7600 out of 43,000) are known from streptomycetes. It has been found that soil streptomycetes are the main source used by bioactive secondary metabolites. However, recently there have been many and varied types of structurally unique and biologically active secondary metabolites found and obtained from marine actinomycetes, including those from the genus Streptomyces. Also, compounds that are synthesized by streptomycetes exhibit extreme chemical diversity. Diverse form made from from simple amino acid derivatives to high molecular weight proteides, and macrolactones from simple eight membered lactones to different condensed macrolactones. Berdy (1974) introduced the first classification scheme for antibiotics referring to the chemical structure. On the basis of Berdy’s scheme, (1996) recognized that both low and high molecular weight compounds from 63 different chemical classes are produced by streptomycetes

Effects Of The Protein Environment On The Spectral Properties Of Tryptophan Radicals In Pseudomonas Aeruginosa Azurin

Many biological electron-transfer reactions involve short-lived tryptophan radicals as key reactive intermediates. While these species are difficult to investigate, the recent photogeneration of a long-lived neutral tryptophan radical in two Pseudomonas aeruginosa azurin mutants (Az48W and ReAz108W) made it possible to characterize the electronic, vibrational, and magnetic properties of such species and their sensitivity to the molecular environment. Indeed, in Az48W the radical is embedded in the hydrophobic core while, in ReAz108W it is solvent-exposed. Here we use density functional theory and multiconfigurational perturbation theory to construct quantum-mechanics/molecular-mechanics models of Az48W* and ReAz108W* capable of reproducing specific features of their observed UV-vis, resonance Raman, and electron paramagnetic resonance spectra. The results show that the models can correctly replicate the spectral changes imposed by the two contrasting hydrophobic and hydrophilic environments. Most importantly, the same models can be employed to disentangle the molecular-level interactions responsible for such changes. It is found that the control of the hydrogen bonding between the tryptophan radical and a single specific surface water molecule in ReAz108W\u27 represents an effective means of spectral modulation. Similarly, a specific electrostatic interaction between the radical moiety and a Val residue is found to control the Az48W* excitation energy. These modulations appear to be mediated by the increase in nitrogen negative charge (and consequent increase in hydrogen bonding) of the spectroscopic D-2 state with respect to the D-0 state of the chromophore. Finally, the same protein models are used to predict the relaxed Az481Ar* and ReAz108W* D-2 structures, showing that the effect of the environment on the corresponding fluorescence maxima must parallel that of Do absorption spectra

Modeling The Fluorescence Of Protein-embedded Tryptophans With Ab Initio Multiconfigurational Quantum Chemistry: The Limiting Cases Of Parvalbumin And Monellin

We show that a quantum-mechanics/molecular-mechanics strategy based on ab initio (i.e., first principle) multiconfigurational perturbation theory can reproduce the spectral properties of a tryptophan residue embedded in the contrasting hydrophobic and hydrophilic environments of parvalbumin and monellin, respectively. We show that the observed absorption and emission energies can be reproduced with a less than 3 kcal mol(-1) error. The analysis of the computed emission energies based on a protein disassembly scheme and protein electrostatic potential mapping allows for a detailed understanding of the factors modulating the tryptophan emission. It is shown that for rnonellin, where the tryptophan is exposed to the solvent, the fluorescence wavelength is controlled not only by the distribution of the point charges of the protein-solvent environment but also by specific hydrogen bonds and, most important, by the environment-induced change in chromophore structure. In contrast, in parvalbumin, where the chromophore is embedded in the protein core, the structure and emission maxima are the same as those of an isolated 3-methylindole fluorophore. Consistently, we find that in parvalbumin the solvation does not change significantly the computed emission energy

Technological breakthrough for energy efficiency in dyes production: bio-synthetic pathway versus chemical process

The industry of dyestuff responds to coloring needs of a wide range of end-use industries, ranging from textile up to construction. In this context research and development activities focused on new technological solutions for low-impact dye synthesis and streamlining manufacturing operations are strategies to be adopted to improve energy efficiency in this sector. Bio-synthetic pathway for dyestuff production represents a high-tech approach that exploits the ability and efficiency of enzymes to catalyse the formation of new dyes. In this study we present the preliminary results of the cradle-to-gate life cycle analysis performed on the biosynthetic production of dyestuff to highlight the potential energy and environmental benefits achievable through the substitution of classical chemical process with enzymatic ones

Spectroscopic characterisation of the naphthalene dioxygenase from Rhodococcus sp. StrainNCIMB12038

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the “peroxide shunt”, an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes

Oxygen Radical Scavenger Activity, EPR, NMR, Molecular Mechanics and Extended-Hückel Molecular Orbital Investigation of the Bis(Piroxicam)Copper(II) Complex

The oxygen radical scavenger activity (ORSA) of [CuII(Pir)2] (HPir = Piroxicam = 4-hydroxy -2- methyl -N-2- pyridyl -2H- 1,2-benzothiazine -3- carboxamide 1,1-dioxide) was determined by chemiluminescence of samples obtained by mixing human neutrophils (from healthy subjects) and [CuII(Pir)2(DMF)2] (DMF = N,N -dimethylformammide) in DMSO/GLY/PBS (2:1:2, v/v) solution (DMSO = dimethylsulfoxide, GLY = 1,2,3-propantriol, PBS = Dulbecco’s buffer salt solution). The ratio of the residual radicals, for the HPir (1.02·10−4M) and [CuII(Pir)2(DMF)2] (1.08·10−5M)/HPir (8.01·10−−5M) systems was higher than 12 (not stimulated) [excess of piroxicam was added (Cu/Pir molar ratio ≈1:10) in order to have most of the metal complexed as bischelate]. In contrast, the ratio of residual radicals for the CuCl2 (1.00·10−5M) and [CuII(Pir)2(DMF)2] (1.08·10−5M)/Hpir (8.01·10−5M)system was 5. The [CuII(Pir)2] compound is therefore a stronger radical scavenger than either HPir or CuCl2. A molecular mechanics (MM) analysis of the gas phase structures of neutral HPir, its zwitterionic (HPir+-) and anionic (Pir-) forms, and some CuII-piroxicam complexes based on X-ray structures allowed calculation of force constants. The most stable structure for HPir has a ZZZ conformation similar to that found in the CuII (and CdII complexes) in the solid state as well as in the gas phase. The structure is stabilized by a strong H bond which involves the N(amide)-H and O(enolic) groups. The MM simulation for the [CuII(Pir)2(DMF)2] complex showed that two high repulsive intramolecular contacts exist between a pyridyl hydrogen atom of one Pir- molecule with the O donor of the other ligand. These interactions activate a transition toward a pseudo-tetrahedral geometry, in the case the apical ligands are removed. On refluxing a suspension of [CuII(Pir)2(DMF)2] in acetone a brown microcystalline solid with the Cu(Pir)2·0.5DMF stoichiometry was in fact prepared. 13C spin-lattice relaxation rates of neutral, zwitterionic and anionic piroxicam, in DMSO solution are explained by the thermal equilibrium between the three most stable structures of the three forms, thus confirming the high quality of the force field. The EPR spectrum of [CuII(Pir)2(DMF)2] (DMSO/GLY, 2:1, v/v, 298 and 110 K) agrees with a N2O2+O2 pseudo-octahedral coordination geometry. The EPR spectrum of [CuII(Pir)2·0.5DMF agrees with a pseudo-tetrahedral coordination geometry. The parameters extracted from the room temperature spectra of the solution phases are in agreement with the data reported for powder and frozen solutions. The extended-Hückel calculations on minimum energy structures of [CuII(Pir)2(DMF)2] and [CuII(Pir)2] (square planar) revealed that the HOMOs have a relevant character of dx2−y2. On the other hand the HOMO of a computer generated structure for [CuII(Pir)2] (pseudo-tetrahedral) has a relevant character of dxy atomic orbital. A dxy orbital is better suited to allow a dπ-pπ interaction to the O2- anion. Therefore this work shows that the anti-inflammatory activity of piroxicam could be due in part to the formation of [CuII(Pir)2] chelates, which can exert a SOD-like activity

Effect of electrode shape and flow conditions on the electrochemical detection with band microelectrodes

In this work, we report the analysis of the electrochemical detection of electroactive species with band microelectrodes that operate under controlled convection. The study focuses on the determination of the collection efficiency of the analyte as a function of inlet flow velocity and microband geometry (inlaid, bumped and recessed), also providing a straightforward method for the theoretical determination of the lower detection limit. The analysis has been carried out by simulating the dimensionless mass transport with the finite element method, delivering the stationary limiting current density. Simulations have been performed on systems consisting of single and double band electrodes to investigate the trail effect on the electrochemical detection. We show that the obtained dimensionless results can be easily turned into dimensional data, providing a tool for the design of devices. The proposed method is general and can easily be extended to systems with different geometry

Basidiomycete DyPs: Genomic diversity, structural–functional aspects, reaction mechanism and environmental significance

The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substratesThis work was supported by the INDOX (KBBE-2013-7-613549; www.indoxproject.eu) European project, the BIO2011-26694 (HIPOP) and CTQ2013-48287 projects of the Spanish Ministry of Economy and Competitiveness (MINECO), and the PRIN 2009-STNWX3 project of the Italian Ministry of Education, University and Research (MIUR). FJR-D thanks a Ramón y Cajal contract of MINECO. The authors thank Verónica Sáez-Jiménez for data on Reactive Blue 5 decolorization by VP and its heme-channel variants.Peer ReviewedPostprint (published version

Spectroscopic characterization of natural melanin from a Streptomyces cyaneofuscatus strain and comparison with melanin enzymatically synthesized by tyrosinase and laccase

An actinobacteria strain was isolated from Algerian Sahara soil and assigned to Streptomyces cyaneofuscatus Pridham et al. 1958 species. This strain was selected for its ability to produce melanin exopigments in liquid and solid media. Melanin synthesis was associated with tyrosinase activity and the enzyme from this strain was isolated and biochemically characterized. Synthetic melanin was then enzymatically produced using the S. cyaneofuscatus Pridham et al. 1958 tyrosinase. As this enzyme showed a higher diphenolase activity, a synthetic melanin from the enzymic oxidation of 3,4-dihydroxyphenylalanine (dopa) was obtained by the use of a Trametes versicolor (L.) Lloyd laccase for comparison. The natural and synthetic pigments were physico-chemically characterized by the use of ultraviolet (UV)-Visible, and Fourier transform infrared (FT-IR) and multifrequency electron paramagnetic resonance (EPR) spectroscopies. All the melanin samples displayed a stable free radical when analyzed by X-band EPR spectroscopy. Once the samples were recorded at Q-band EPR, a copolymer derived from a mixture of different constituents was evident in the natural melanin. All radical species were analyzed and discussed. The use of water-soluble melanin naturally produced by S. cyaneofuscatus Pridham et al. 1958 represents a new biotechnological alternative to commercial insoluble pigments