Understanding the spatio-temporal behaviour of the sunflower crop for subfield areas delineation using Sentinel-2 NDVI time-series images in an organic farming system.

The study investigates the suitability of time series Sentinel-2 NDVI-derived maps for the subfield detection of a sunflower crop cultivated in an organic farming system. The aim was to understand the spatio-temporal behaviour of subfield areas identified by the K-means algorithm from NDVI maps obtained from satellite images and the ground yield data variability to increase the efficiency of delimiting management zones in an organic farming system. Experiments were conducted on a surface of 29 ha. NDVI time series derived from Sentinel-2 images and k-means algorithm for rapidly delineating the sunflower subfield areas were used. The crop achene yields in the whole field ranged from 1.3 to 3.77 t ha-1 with a significant within-field spatial variability. The cluster analysis of hand-sampled data showed three subfields with achene yield mean values of 3.54 t ha-1 (cluster 1), 2.98 t ha-1 (cluster 2), and 2.07 t ha-1 (Cluster 3). In the cluster analysis of NDVI data, the k-means algorithm has early delineated the subfield crop spatial and temporal yield variability. The best period for identifying subfield areas starts from the inflorescences development stage to the development of the fruit stage. Analyzing the NDVI subfield areas and yield data, it was found that cluster 1 covers an area of 42.4% of the total surface and 50% of the total achene yield; cluster 2 covers 35% of both surface and yield. Instead, the surface of cluster 3 covers 22.2% of the total surface with 15% of achene yield. K-means algorithm derived from Sentinel-2 NDVI images delineates the sunflower subfield areas. Sentinel-2 images and k-means algorithms can improve an efficient assessment of subfield areas in sunflower crops. Identifying subfield areas can lead to site-specific long-term agronomic actions for improving the sustainable intensification of agriculture in the organic farming system.

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus: Pushing the Limits of Nanopore Sequencing.

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

Study on analytical characteristics of Nicotiana tabacum L., cv. Solaris biomass for potential uses in nutrition and biomethane production.

In order to limit the smoking tobacco sector crisis, a new non-GMO Nicotiana tabacum L. cv. Solaris was proposed as oil seed crop. Residues of oil extraction were successfully used in swine nutrition. The aim of this study was to explore the full potential of this innovative tobacco cultivar as multitasking feedstock non interfering with the food chain. In the triennium 2016-2018, samples from whole plant, inflorescence and stem-leaf biomass were collected in three experimental sites and analysed for chemical constituents, including fibre fractions, sugars and starch, macro-minerals and total alkaloids. The KOH soluble protein content and the amino-acid profile were also investigated as well as the biochemical methane potential. All the analyses were performed according to official methods and results were compared with values reported in literature for conventional lignocellulosic crops and agro-industry residues. The average protein content, ranging from 16.01 to 18.98 g 100 g-1 dry matter respectively for stem-leaf and whole plant samples, and their amino-acid profile are consistent with values reported for standard grass plant. These findings suggest the potential use of cv. Solaris in industrial food formulations. Moreover, considering the average content of both fibre available for fermentations (72.6% of Neutral Detergent Fibre) and oils and fats (7.92 g 100 g-1 dry matter), the whole plant biomass of cv. Solaris showed good attitude to anaerobic fermentation, confirmed by the biochemical methane potential of whole plant (168 Nm3 t-1 organic matter). Similarly, results allow to define the cv. Solaris biomass as a good quality forage apt to ensiling for its chemical composition. The low total alkaloids content of cv. Solaris, in average 0.3 g 100 g-1 dry matter, was previously reported not to affect growth performances and welfare traits of dairy heifers. These are the first results showing the multitasking potential use of cv. Solaris biomass, that could allow the recovery of tobacco cultivation know-how especially in marginal areas.

NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension.

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-ß (SMAD-TGF-ß) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-ß ligand trap and, thus, was not contingent on TGF-ß signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-ß-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.

Cpipe: a comprehensive computational platform for sequence and structure-based analyses of Cysteine residues.

SUMMARY: Due to their chemical plasticity, Cysteine residues (Cys) can serve many different functions. Identification and classification of reactive Cys isn't a trivial job: currently, no available tool exists for an all-round, comprehensive (inclusive of all different functional types) analysis of Cys; herein we present a computational platform called Cp i pe, dedicated to this task: it implements state-of-the art protocols, elaborating and displaying a wealth of information, sufficiently orthogonal to allow a thorough evaluation of all major aspects of Cys reactivity. AVAILABILITY AND IMPLEMENTATION: Cp i pe is implemented in Python and freely available at http://cpipe.explora-biotech.com/cpipe/start.py . All major browsers are supported. CONTACT: s.marino@explora-biotech.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Variation in Ecophysiological Traits and Drought Tolerance of Beech (Fagus sylvatica L.) Seedlings from Different Populations.

Frequency and intensity of heat waves and drought events are expected to increase in Europe due to climate change. European beech (Fagus sylvatica L.) is one of the most important native tree species in Europe. Beech populations originating throughout its native range were selected for common-garden experiments with the aim to determine whether there are functional variations in drought stress responses among different populations. One-year old seedlings from four to seven beech populations were grown and drought-treated in a greenhouse, replicating the experiment at two contrasting sites, in Italy (Mediterranean mountains) and Germany (Central Europe). Experimental findings indicated that: (1) drought (water stress) mainly affected gas exchange describing a critical threshold of drought response between 30 and 26% SWA for photosynthetic rate and Ci/Ca, respectively; (2) the Ci to Ca ratio increased substantially with severe water stress suggesting a stable instantaneous water use efficiency and an efficient regulation capacity of water balance achieved by a tight stomatal control; (3) there was a different response to water stress among the considered beech populations, differently combining traits, although there was not a well-defined variability in drought tolerance. A combined analysis of functional and structural traits for detecting stress signals in beech seedlings is suggested to assess plant performance under limiting moisture conditions and, consequently, to estimate evolutionary potential of beech under a changing environmental scenario.

Cy-preds: An algorithm and a web service for the analysis and prediction of cysteine reactivity.

Cysteine (Cys) is a critically important amino acid, serving a variety of functions within proteins including structural roles, catalysis, and regulation of function through post-translational modifications. Predicting which Cys residues are likely to be reactive is a very sought after feature. Few methods are currently available for the task, either based on evaluation of physicochemical features (e.g., pKa and exposure) or based on similarity with known instances. In this study, we developed an algorithm (named HAL-Cy) which blends previous work with novel implementations to identify reactive Cys from nonreactive. HAL-Cy present two major components: (i) an energy based part, rooted on the evaluation of H-bond network contributions and (ii) a knowledge based part, composed of different profiling approaches (including a newly developed weighting matrix for sequence profiling). In our evaluations, HAL-Cy provided significantly improved performances, as tested in comparisons with existing approaches. We implemented our algorithm in a web service (Cy-preds), the ultimate product of our work; we provided it with a variety of additional features, tools, and options: Cy-preds is capable of performing fully automated calculations for a thorough analysis of Cys reactivity in proteins, ranging from reactivity predictions (e.g., with HAL-Cy) to functional characterization. We believe it represents an original, effective, and very useful addition to the current array of tools available to scientists involved in redox biology, Cys biochemistry, and structural bioinformatics.

Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks.

S-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.

Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins.

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.

Selenoprotein S is involved in maintenance and transport of multiprotein complexes.

SelS (Selenoprotein S) is a selenocysteine-containing protein with roles in ER (endoplasmic reticulum) function and inflammation. It has been implicated in ERAD (ER-associated protein degradation), and clinical studies revealed an association of its promoter polymorphism with cytokine levels and human diseases. However, the pathways and interacting proteins that could shed light on pathogenesis of SelS-associated diseases have not been studied systematically. We performed a large-scale affinity isolation of human SelS and its mutant forms and analysed the proteins that interact with them. All previously known SelS targets and nearly two hundred additional proteins were identified that were remarkably enriched for various multiprotein complexes. Subsequent chemical cross-linking experiments identified the specific interacting sites in SelS and its several targets. Most of these interactions involved coiled-coil domains. The data suggest that SelS participates in intracellular membrane transport and maintenance of protein complexes by anchoring them to the ER membrane.

Protein flexibility and cysteine reactivity: influence of mobility on the H-bond network and effects on pKa prediction.

Thanks to its chemical plasticity, cysteine (Cys) is a very versatile player in proteins. A major determinant of Cys reactivity is pKa: the ability to predict it is deemed critical in redox bioinformatics. I considered different computational methods for pKa predictions and ultimately applied one (propka, ppka1) to various datasets; for all residues I assessed the effect of (1) hydrogen bonding, electrostatics and solvation on predictions and (2) protein mobility on pKa variability. Particularly for Cys, exposure and H-bond contributions heavily dictated propka predictions. The prominence of H-bond contributions was previously reported: this may explain the effectiveness of ppka1 (with Cys, tested in a benchmark). However ppka1 was also very sensitive to protein mobility; I assessed the effects of mobility on particularly large (compared to previous studies) datasets of structural ensembles; I found that exposed Cys presented the highest pKa variability, ascribable to correspondingly high H-bond fluctuations associated with protein flexibility. The benefit of including protein dynamics in pKa predictions was previously proposed, but empirical methods were never tested in this sense; instead, giving their outstanding speed, they could lend particularly well to this purpose. I devised a strategy combining short range molecular dynamics with ppka1; the protocol aimed to mitigate high ppka1 variability by including a "statistical view" of fast conformational changes. Tested in a benchmark, the strategy lead to improved performances. These results provide new insights on Cys bioinformatics (pKa prediction protocols) and Cys biology (effect of mobility on exposed Cys properties).

Selenocysteine in thiol/disulfide-like exchange reactions.

SIGNIFICANCE: Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. RECENT ADVANCES: The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. CRITICAL ISSUES: We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. FUTURE DIRECTIONS: It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

Glomerulitis and endothelial cell enlargement in C4d+ and C4d- acute rejections of renal transplant patients.

In acute rejection after renal transplant, glomerulitis is characterized by mononuclear cells in glomerular capillaries and endothelial cell enlargement. In association with C4d deposition in peritubular capillaries, glomerulitis is a feature of acute antibody-mediated rejection. Prognosis in C4d(+) rejection is poorer than in C4d(-) rejection. We measured the glomerular endothelial cell area in C4d(+) and C4d(-) acute rejections by morphometry. In 90 acute rejection biopsies, glomerulitis was present in 36 cases (group G) and absent in 54 (group G0). In biopsies without rejections and in C4d(-) biopsies of group G0, glomerular endothelial cell area was not significantly different. In C4d(-) and C4d(+) biopsies of group G, the area in inflamed glomeruli was greater than that in C4d(-) biopsies of group G0 (P < .02 and P < .006, respectively). In C4d(+) biopsies of group G0, it was, unexpectedly, greater than in C4d(-) biopsies of group G (P < .01). Circulating posttransplant anti-human leukocyte antigen class I and class II antibodies correlated with increased endothelial cell area (P < .02). Glomerulitis was associated with diffuse C4d deposition (odds ratio [OR], 4.27; P < .004); C4d deposition was associated with steroid resistance (OR, 4.97; P < .002). Only in C4d(+) rejections did the presence of glomerulitis increase this association (OR, 9.17; P < .02). In conclusion, we quantified an increase of endothelial cell area in glomerulitis of C4d(+) and C4d(-) acute rejections (group G). An increase of this area in C4d(+) biopsies without glomerulitis (group G0) suggests complement-mediated damage in the absence of mononuclear cell margination.

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding.

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.

Analysis and functional prediction of reactive cysteine residues.

Cys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pK(a), to algorithms for functional prediction of different types of Cys in proteins.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat.

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies.

Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction.

A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines.

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.

Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols.

Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2-ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a π-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols.